Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-lysine

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Peschel, A.; Jack, R.W.; Otto, M.; Collins, L.V.; Staubitz, P.; Nicholson, G.; Kalbacher, H.; Nieuwenhuizen, W.F.; Jung, G.; Tarkowski, A.; Kessel, K.P.M. van; Strijp, J.A.G. van

2001-01-01

Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance

Increased expression and levels of human β defensins (hBD2 and hBD4 in adults with dental caries

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Girolamo Jose Barrera

2013-09-01

Full Text Available Introduction: Defensins are small anti-microbial peptides produced by epithelial cells. These peptides have a broad range of actions against microorganisms, including Gram-positive and Gram-negative bacteria.Human defensins are classifi ed into two subfamilies, the α-, and β- defensins, which differ in their distribution of disulphide bonds between the six conserved cysteine residues. Defensins are found in salivaand others compartments of the body. Human β defensins 2 (hBD2, beta defensins 4 (hBD4 and alpha defensins 4 (hNP4 in saliva may contributes to vulnerability or resistance to caries. This study aimed to determine a possible correlation between caries and levels of defensins measuring the expression in gingival tissue and concentrations in saliva samples.Methods: Oral examinations were performed on 100 adults of both genders (18-30 years old, and unstimulated whole saliva was collected for immunoassays of the three peptides and for the salivary pH, buffercapacity, protein, and peroxidase activity. mRNA levels of defensins in gingival sample were assessed by semi-quantitative RT-PCR technique.Results: The median salivary levels of hBD2 and hBD4 were 1.88 μg/ml and 0.86 μg/ml respectively for the caries-free group (n=44 and 7.26 μ/ml (hBD2 and 4.25 μg/ml (hBD4 for all subjects with evidenceof caries (n=56. There was no difference in the levels of hNP4, salivary pH, and proteins between groups, however the peroxidase activity and buffer capacity (interval 6.0-5.0 were reduced in caries group. Transcriptional levels of hBD2 and hBD4 did correlate with caries experience, the mRNA expression of hBD2 and hBD4 were signifi cantly higher in patients with caries than in patients with no-caries (p Conclusion: We conclude that high salivary levels and expression of beta defensins, low peroxidase activity and buffer capacity may represent a biological response of oral tissue to caries. Our observation couldlead to new ways to prevent caries

Increased expression and levels of human β defensins (hBD2 and hBD4 in adults with dental caries

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Girolamo Jose Barrera

2013-09-01

Full Text Available Introduction: Defensins are small anti-microbial peptides produced by epithelial cells. These peptides have a broad range of actions against microorganisms, including Gram-positive and Gram-negative bacteria.Human defensins are classifi ed into two subfamilies, the α-, and β- defensins, which differ in their distribution of disulphide bonds between the six conserved cysteine residues. Defensins are found in salivaand others compartments of the body. Human β defensins 2 (hBD2, beta defensins 4 (hBD4 and alpha defensins 4 (hNP4 in saliva may contributes to vulnerability or resistance to caries. This study aimed to determine a possible correlation between caries and levels of defensins measuring the expression in gingival tissue and concentrations in saliva samples.Methods: Oral examinations were performed on 100 adults of both genders (18-30 years old, and unstimulated whole saliva was collected for immunoassays of the three peptides and for the salivary pH, buffercapacity, protein, and peroxidase activity. mRNA levels of defensins in gingival sample were assessed by semi-quantitative RT-PCR technique.Results: The median salivary levels of hBD2 and hBD4 were 1.88 μg/ml and 0.86 μg/ml respectively for the caries-free group (n=44 and 7.26 μ/ml (hBD2 and 4.25 μg/ml (hBD4 for all subjects with evidenceof caries (n=56. There was no difference in the levels of hNP4, salivary pH, and proteins between groups, however the peroxidase activity and buffer capacity (interval 6.0-5.0 were reduced in caries group. Transcriptional levels of hBD2 and hBD4 did correlate with caries experience, the mRNA expression of hBD2 and hBD4 were signifi cantly higher in patients with caries than in patients with no-caries (p < 0.01.Conclusion: We conclude that high salivary levels and expression of beta defensins, low peroxidase activity and buffer capacity may represent a biological response of oral tissue to caries. Our observation couldlead to new ways to prevent

Isolation and Properties of Floral Defensins from Ornamental Tobacco and Petunia1

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Lay, Fung T.; Brugliera, Filippa; Anderson, Marilyn A.

2003-01-01

The flowers of the solanaceous plants ornamental tobacco (Nicotiana alata) and petunia (Petunia hybrida) produce high levels of defensins during the early stages of development. In contrast to the well-described seed defensins, these floral defensins are produced as precursors with C-terminal prodomains of 27 to 33 amino acids in addition to a typical secretion signal peptide and central defensin domain of 47 or 49 amino acids. Defensins isolated from N. alata and petunia flowers lack the C-terminal domain, suggesting that it is removed during or after transit through the secretory pathway. Immunogold electron microscopy has been used to demonstrate that the N. alata defensin is deposited in the vacuole. In addition to the eight canonical cysteine residues that define the plant defensin family, the two petunia defensins have an extra pair of cysteines that form a fifth disulfide bond and hence define a new subclass of this family of proteins. Expression of the N. alata defensin NaD1 is predominantly flower specific and is most active during the early stages of flower development. NaD1 transcripts accumulate in the outermost cell layers of petals, sepals, anthers, and styles, consistent with a role in protection of the reproductive organs against potential pathogens. The floral defensins inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro, providing further support for a role in protection of floral tissues against pathogen invasion. PMID:12644678

Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants.

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de Zélicourt, Axel; Letousey, Patricia; Thoiron, Séverine; Campion, Claire; Simoneau, Philippe; Elmorjani, Khalil; Marion, Didier; Simier, Philippe; Delavault, Philippe

2007-08-01

Plant defensins are small basic peptides of 5-10 kDa and most of them exhibit antifungal activity. In a sunflower resistant to broomrape, among the three defensin encoding cDNA identified, SF18, SD2 and HaDef1, only HaDef1 presented a preferential root expression pattern and was induced upon infection by the root parasitic plant Orobanche cumana. The amino acid sequence deduced from HaDef1 coding sequence was composed of an endoplasmic reticulum signal sequence of 28 amino acids, a standard defensin domain of 50 amino-acid residues and an unusual C-terminal domain of 30 amino acids with a net positive charge. A 5.8 kDa recombinant mature Ha-DEF1 corresponding to the defensin domain was produced in Escherichia coli and was purified by means of a two-step chromatography procedure, Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography. Investigation of in vitro antifungal activity of Ha-DEF1 showed a strong inhibition on Saccharomyces cerevisiae growth linked to a membrane permeabilization, and a morphogenetic activity on Alternaria brassicicola germ tube development, as already reported for some other plant defensins. Bioassays also revealed that Ha-DEF1 rapidly induced browning symptoms at the radicle apex of Orobanche seedlings but not of another parasitic plant, Striga hermonthica, nor of Arabidopsis thaliana. FDA vital staining showed that these browning areas corresponded to dead cells. These results demonstrate for the first time a lethal effect of defensins on plant cells. The potent mode of action of defensin in Orobanche cell death and the possible involvement in sunflower resistance are discussed.

Defensins in periodontal health

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Taran Bedi

2015-01-01

Full Text Available Defensins are abundant and widely distributed peptides in human and animal tissues that are involved in host defence. Defensins not only have the ability to strengthen the innate immune system but can also enhance the adaptive immune system by chemotaxis of monocytes, T-lymphocytes, dendritic cells and mast cells to the infection site. Defensins also improves the capacity of macrophage phagocytosis. A greater understanding of how these peptides act in the healthy, gingivitis and periodontitis conditions would definitely open new opportunities for identification, prevention and treatment of periodontal diseases. This discussion focuses on recent studies about biological function of defensins in human diseases and animal models.

Expression of apoplast-targeted plant defensin MtDef4.2 confers resistance to leaf rust pathogen Puccinia triticina but does not affect mycorrhizal symbiosis in transgenic wheat.

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Kaur, Jagdeep; Fellers, John; Adholeya, Alok; Velivelli, Siva L S; El-Mounadi, Kaoutar; Nersesian, Natalya; Clemente, Thomas; Shah, Dilip

2017-02-01

Rust fungi of the order Pucciniales are destructive pathogens of wheat worldwide. Leaf rust caused by the obligate, biotrophic basidiomycete fungus Puccinia triticina (Pt) is an economically important disease capable of causing up to 50 % yield losses. Historically, resistant wheat cultivars have been used to control leaf rust, but genetic resistance is ephemeral and breaks down with the emergence of new virulent Pt races. There is a need to develop alternative measures for control of leaf rust in wheat. Development of transgenic wheat expressing an antifungal defensin offers a promising approach to complement the endogenous resistance genes within the wheat germplasm for durable resistance to Pt. To that end, two different wheat genotypes, Bobwhite and Xin Chun 9 were transformed with a chimeric gene encoding an apoplast-targeted antifungal plant defensin MtDEF4.2 from Medicago truncatula. Transgenic lines from four independent events were further characterized. Homozygous transgenic wheat lines expressing MtDEF4.2 displayed resistance to Pt race MCPSS relative to the non-transgenic controls in growth chamber bioassays. Histopathological analysis suggested the presence of both pre- and posthaustorial resistance to leaf rust in these transgenic lines. MtDEF4.2 did not, however, affect the root colonization of a beneficial arbuscular mycorrhizal fungus Rhizophagus irregularis. This study demonstrates that the expression of apoplast-targeted plant defensin MtDEF4.2 can provide substantial resistance to an economically important leaf rust disease in transgenic wheat without negatively impacting its symbiotic relationship with the beneficial mycorrhizal fungus.

A Scorpion Defensin BmKDfsin4 Inhibits Hepatitis B Virus Replication in Vitro

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Zhengyang Zeng

2016-04-01

Full Text Available Hepatitis B virus (HBV infection is a major worldwide health problem which can cause acute and chronic hepatitis and can significantly increase the risk of liver cirrhosis and primary hepatocellular carcinoma (HCC. Nowadays, clinical therapies of HBV infection still mainly rely on nucleotide analogs and interferons, the usage of which is limited by drug-resistant mutation or side effects. Defensins had been reported to effectively inhibit the proliferation of bacteria, fungi, parasites and viruses. Here, we screened the anti-HBV activity of 25 scorpion-derived peptides most recently characterized by our group. Through evaluating anti-HBV activity and cytotoxicity, we found that BmKDfsin4, a scorpion defensin with antibacterial and Kv1.3-blocking activities, has a comparable high inhibitory rate of both HBeAg and HBsAg in HepG2.2.15 culture medium and low cytotoxicity to HepG2.2.15. Then, our experimental results further showed that BmKDfsin4 can dose-dependently decrease the production of HBV DNA and HBV viral proteins in both culture medium and cell lysate. Interestingly, BmKDfsin4 exerted high serum stability. Together, this study indicates that the scorpion defensin BmKDfsin4 also has inhibitory activity against HBV replication along with its antibacterial and potassium ion channel Kv1.3-blocking activities, which shows that BmKDfsin4 is a uniquely multifunctional defensin molecule. Our work also provides a good molecule material which will be used to investigate the link or relationship of its antiviral, antibacterial and ion channel–modulating activities in the future.

Characterization of Cimex lectularius (bedbug) defensin peptide and its antimicrobial activity against human skin microflora.

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Kaushal, Akanksha; Gupta, Kajal; van Hoek, Monique L

2016-02-19

Antimicrobial peptides are components of both vertebrate and invertebrate innate immune systems that are expressed in response to exposure to bacterial antigens. Naturally occurring antimicrobial peptides from evolutionarily ancient species have been extensively studied and are being developed as potential therapeutics against antibiotic resistant microorganisms. In this study, a putative Cimex lectularius (bedbug, CL) defensin is characterized for its effectiveness against human skin flora including Gram-negative and Gram-positive bacteria. The bedbug defensin (CL-defensin), belonging to family of insect defensins, is predicted to have a characteristic N-terminal loop, an α-helix, and an antiparallel β-sheet, which was supported by circular dichroism spectroscopy. The defensin was shown to be antimicrobial against Gram-positive bacteria commonly found on human skin (Micrococcus luteus, Corynebacterium renale, Staphylococcus aureus and Staphylococcus epidermidis); however, it was ineffective against common skin Gram-negative bacteria (Pseudomonas aeruginosa and Acinetobacter baumannii) under low-salt conditions. CL-defensin was also effective against M. luteus and C. renale in high-salt (MIC) conditions. Our studies indicate that CL-defensin functions by depolarization and pore-formation in the bacterial cytoplasmic membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

Transfer and expression of the rabbit defensin NP-1 gene in lettuce (Lactuca sativa).

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Song, D; Xiong, X; Tu, W F; Yao, W; Liang, H W; Chen, F J; He, Z Q

2017-01-23

Lettuce (Lactuca sativa L.) is an annual plant of the daisy family, Asteraceae, with high food and medicinal value. However, the crop is susceptible to several viruses that are transmitted by aphids and is highly vulnerable to post-harvest diseases, as well as insect and mammal pests and fungal and bacterial diseases. Here, the rabbit defensin gene NP-1 was transferred into lettuce by Agrobacterium-mediated transformation to obtain a broad-spectrum disease-resistant lettuce. Transgenic lettuce plants were selected and regenerated on selective media. The presence of the NP-1 gene in these plants was confirmed by western blot analyses. Resistance tests revealed native defensin NP-1 expression conferred partial resistance to Bacillus subtilis and Pseudomonas aeruginosa, which suggests new possibilities for lettuce disease resistance.

Beta-defensin-2 protein is a serum biomarker for disease activity in psoriasis and reaches biologically relevant concentrations in lesional skin.

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Patrick A M Jansen

Full Text Available BACKGROUND: Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2 is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis. METHODOLOGY/PRINCIPAL FINDINGS: We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects. CONCLUSIONS/SIGNIFICANCE: Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases.

Beta-defensin genomic copy number is not a modifier locus for cystic fibrosis

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Burgess Juliana

2005-12-01

Full Text Available Abstract Human beta-defensin 2 (DEFB4, also known as DEFB2 or hBD-2 is a salt-sensitive antimicrobial protein that is expressed in lung epithelia. Previous work has shown that it is encoded in a cluster of beta-defensin genes at 8p23.1, which varies in copy number between 2 and 12 in different individuals. We determined the copy number of this locus in 355 patients with cystic fibrosis (CF, and tested for correlation between beta-defensin cluster genomic copy number and lung disease associated with CF. No significant association was found.

Human alpha-defensin-1 protects cells from intoxication with Clostridium perfringens iota toxin.

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Fischer, Stephan; Popoff, Michel R; Barth, Holger

2018-03-01

Iota toxin is produced by Clostridium perfringens type E strains and associated with diarrhea in cattle and lambs. This binary protein toxin comprises the enzyme component iota a (Ia), which ADP-ribosylates G-actin, and the separate transport component iota b (Ib), which delivers Ia into the cytosol of target cells. Ib binds to cell receptors and forms biologically active toxin complexes with Ia, which cause rounding of adherent cells due to the destruction of the actin cytoskeleton. Here, we report that the human peptide α-defensin-1 protects cultured cells including human colon cells from intoxication with iota toxin. In contrast, the related ß-defensin-1 had no effect, indicating a specific mode of action. The α-defensin-1 did not inhibit ADP-ribosylation of actin by Ia in vitro. Pretreatment of Ib with α-defensin-1 prior to addition of Ia prevented intoxication. Additionally, α-defensin-1 protected cells from cytotoxic effects mediated by Ib in the absence of Ia, implicating that α-defensin-1 interacts with Ib to prevent the formation of biologically active iota toxin on cells. In conclusion, the findings contribute to a better understanding of the functions of α-defensin-1 and suggest that this human peptide might be an attractive starting point to develop novel pharmacological options to treat/prevent diseases associated with iota toxin-producing Clostridium perfringens strains.

Regulation of gene expression for defensins and lipid transfer protein in Scots pine seedlings by necrotrophic pathogen Alternaria alternata (Fr.

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Hrunyk Nataliya

2017-06-01

Full Text Available Damping-off disease in pine seedling, caused by fungi and oomycetes (Fusarium, Alternaria, Botrytis, Phytophthora and other species, is one of the most dangerous diseases in conifer nurseries and greenhouses worldwide. Alternaria alternata is a necrotrophic pathogen, which causes early blight in higher plants and results in massive economic losses in agro-industry as well as in forestry. Pine seedlings that lack strong lignificated and suberized cell walls at early stages of their growth are vulnerable to damping-off disease. So, triggering the synthesis of antimicrobial compounds, such as phytoalexins, anticipins and pathogenesis-related (PR proteins, is the main defense strategy to confine pathogens at early stages of pine ontogenesis. Defensins and lipid transfer proteins are members of two PR-protein families (PR-12 and PR-14 respectively and possess antimicrobial activities in vitro through contact toxicity, and the involvement in defense signalling. In this work, we describe the changes in the expression levels of four defensin genes and lipid transfer protein in Scots pine seedlings infected with A. alternata. The expression levels of PsDef1 and PsDef2 increased at 48 h.p.i. (hours post inoculation. The levels of PsDef4 transcripts have increased after 6 and 24 hours. Notably, at 48 h.p.i., the level of PsDef4 transcripts was decreased by 1.2 times compared to control. The level of PsDef3 transcripts was reduced at all three time points. On the other hand, the level of PsLTP1 transcripts increased at 6 h and 48 h.p.i.; while at 24 h.p.i., it decreased by 20% when compared to the control sample. Our results suggest that defensins and lipid transfer protein are involved in the defense response of young Scots pine to necrotrophic pathogen. Thus, those genes can be used as the molecular markers in forestry selection and development of the ecologically friendly remedies for coniferous seedlings cultivation in greenhouses and nurseries.

A crucial role of paralogous β-defensin genes in the Chinese alligator innate immune system revealed by the first determination of a Crocodilia defensin cluster.

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Tang, Ke-Yi; Wang, Xin; Wan, Qiu-Hong; Fang, Sheng-Guo

2018-04-01

The β-defensin, one of the antimicrobial peptides (AMPs), is a significant component of the innate immune with a broad range of antimicrobial activities. Differing from the widely-studied mammals and birds, limited information about β-defensins has been reported in reptiles, especially in crocodilians. As a same ancient species as dinosaurs and the most endangered species of 23 crocodilians, the survival of Chinese alligator (Alligator sinensis) means a powerful immune system and possible involvement of AMPs in its immune resistance. In this study, we identified 20 novel Alligator sinensisβ-defensin genes (AsBDs) from a 390 kb region using bioinformatic and experimental approaches, and successfully distinguished six orthologous AsBDs to birds and nine paralogous AsBDs undergoing gene duplication events. The amino acid alignment shows that the AsBD paralogs, like α-defensins, encode a significantly longer pro-piece comparing with the orthologs. The calculation of non-synonymous (d N ) and synonymous (d S ) substitutions in the mature peptide reveals that the AsBD paralogs experience a significantly higher selective pressure (d N /d S ) than the orthologs, but a similar evolutionary force to α-defensins. The gene expression result indicates that the AsBD paralogs have a significantly higher expression level than the orthologos in gastrointestinal tract where the host is vulnerable to enteric pathogenic bacteria, as observed in α-defensins. These three pieces of evidence demonstrate that the AsBD paralogs do play an important role in maintaining long-term survival of this endangered reptile. Thus, this survey of AsBDs on the genomic structure, evolutionary characteristics, and expression pattern provides a genetic and immunological foundation for further investigating their antimicrobial function and alternative antibiotics potentiality. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Alpha-defensins 1-3 release by dendritic cells is reduced by estrogen

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Sperling Rhoda

2011-08-01

Full Text Available Abstract Background During pregnancy the immune system of the mother must protect any activation that may negatively affect the fetus. Changes in susceptibility to infection as well as resolution of some autoimmune disorders represent empirical evidence for pregnancy related alterations in immunity. Sex hormones reach extremely high levels during pregnancy and have been shown to have direct effects on many immune functions including the antiviral response of dendritic cells. Among the immunologically active proteins secreted by monocyte derived DCs (MDDC are the alpha-defensins 1-3. This family of cationic antimicrobial peptides has a broad spectrum of microbicidal activity and has also been shown to link innate to adaptive immunity by attracting T cells and immature DCs, which are essential for initiating and polarizing the immune response. Methods We compare culture-generated monocyte derived DCs (MDDCs with directly isolated myeloid dendritic cells (mDCs and plasmacytoid dendritic cells (pDCs and measure their alpha-defensins 1-3 secretion by ELISA both, in basal situations and after hormone (E2 or PG treatments. Moreover, using a cohort of pregnant women we isolated mDCs from blood and also measure the levels of these anti-microbial peptides along pregnancy. Results We show that mDCs and pDCs constitutively produce alpha-defensins 1-3 and at much higher levels than MDDCs. Alpha-defensins 1-3 production from mDCs and MDDCs but not pDCs is inhibited by E2. PG does not affect alpha-defensins 1-3 in any of the populations. Moreover, alpha-defensins 1-3 production by mDCs was reduced in the later stages of pregnancy in 40% of the patients. Conclusions Here, we demonstrate that mDCs and pDCs secrete alpha-defensins 1-3 and present a novel effect of E2 on the secretion of alpha-defensins 1-3 by dendritic cells.

Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus

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French Frank S

2006-02-01

Full Text Available Abstract Background beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation. Methods In silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118–123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities. Results Novel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10–60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity. Conclusion Rat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis.

Antibacterial activity of defensin PaDef from avocado fruit (Persea americana var. drymifolia) expressed in endothelial cells against Escherichia coli and Staphylococcus aureus.

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Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Suárez-Rodríguez, Luis M; Salgado-Garciglia, Rafael; Rodríguez-Zapata, Luis C; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2013-01-01

Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP) represent attractive control agents. Mexican avocado (Persea americana var. drymifolia) is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp) encoding a protein (78 aa) homologous with plant defensins (>80%). We expressed the defensin PaDef cDNA (pBME3) in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 μg/mL of total protein from clones (>55%). Also, S. aureus viability was inhibited from 50 μg/mL total protein (27-38%) but was more evident at 100 μg/mL (52-65%). This inhibition was higher than the effect showed by polyclonal population (~23%). Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

Antibacterial Activity of Defensin PaDef from Avocado Fruit (Persea americana var. drymifolia Expressed in Endothelial Cells against Escherichia coli and Staphylococcus aureus

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Jaquelina Julia Guzmán-Rodríguez

2013-01-01

Full Text Available Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP represent attractive control agents. Mexican avocado (Persea americana var. drymifolia is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp encoding a protein (78 aa homologous with plant defensins (>80%. We expressed the defensin PaDef cDNA (pBME3 in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 μg/mL of total protein from clones (>55%. Also, S. aureus viability was inhibited from 50 μg/mL total protein (27–38% but was more evident at 100 μg/mL (52–65%. This inhibition was higher than the effect showed by polyclonal population (~23%. Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

Candida Infections and Human Defensins.

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Polesello, Vania; Segat, Ludovica; Crovella, Sergio; Zupin, Luisa

2017-01-01

Candida species infections are an important worldwide health issue since they do not only affect immunocompromised patients but also healthy individuals. The host developed different mechanisms of protection against Candida infections; specifically the immune system and the innate immune response are the first line of defence. Defensis are a group of antimicrobial peptides, components of the innate immunity, produced at mucosal level and known to be active against bacteria, virus but also fungi. The aim of the current work was to review all previous studies in literature that analysed defensins in the context of Candida spp. infections, in order to investigate and clarify the exact mechanisms of defensins anti-fungal action. Several studies were identified from 1985 to 2017 (9 works form years 1985 to 1999, 44 works ranging from 2000 to 2009 and 35 from 2010 to 2017) searched in two electronic databases (PubMed and Google Scholar). The main key words used for the research were "Candida", "Defensins"," Innate immune system","fungi". The findings of the reviewed studies highlight the pivotal role of defensins antimicrobial peptides in the immune response against Candida infections, since they are able to discriminate host cell from fungi: defensins are able to recognize the pathogens cell wall (different in composition from the human ones), and to disrupt it through membrane permeabilization. However, further research is needed to explain completely defensins' mechanisms of action to fight C. albicans (and other Candida spp.) infections, being the information fragmentary and only in part elucidated. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Diversification of defensins and NLRs in Arabidopsis species by different evolutionary mechanisms.

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Mondragón-Palomino, Mariana; Stam, Remco; John-Arputharaj, Ajay; Dresselhaus, Thomas

2017-12-15

Genes encoding proteins underlying host-pathogen co-evolution and which are selected for new resistance specificities frequently are under positive selection, a process that maintains diversity. Here, we tested the contribution of natural selection, recombination and transcriptional divergence to the evolutionary diversification of the plant defensins superfamily in three Arabidopsis species. The intracellular NOD-like receptor (NLR) family was used for comparison because positive selection has been well documented in its members. Similar to defensins, NLRs are encoded by a large and polymorphic gene family and many of their members are involved in the immune response. Gene trees of Arabidopsis defensins (DEFLs) show a high prevalence of clades containing orthologs. This indicates that their diversity dates back to a common ancestor and species-specific duplications did not significantly contribute to gene family expansion. DEFLs are characterized by a pervasive pattern of neutral evolution with infrequent positive and negative selection as well as recombination. In comparison, most NLR alignment groups are characterized by frequent occurrence of positive selection and recombination in their leucine-rich repeat (LRR) domain as well negative selection in their nucleotide-binding (NB-ARC) domain. While major NLR subgroups are expressed in pistils and leaves both in presence or absence of pathogen infection, the members of DEFL alignment groups are predominantly transcribed in pistils. Furthermore, conserved groups of NLRs and DEFLs are differentially expressed in response to Fusarium graminearum regardless of whether these genes are under positive selection or not. The present analyses of NLRs expands previous studies in Arabidopsis thaliana and highlights contrasting patterns of purifying and diversifying selection affecting different gene regions. DEFL genes show a different evolutionary trend, with fewer recombination events and significantly fewer instances of

Antiplasmodial activity is an ancient and conserved feature of tick defensins

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Alejandro Cabezas-Cruz

2016-10-01

Full Text Available Ancestral sequence reconstruction has been widely used to test evolution-based hypotheses. The genome of the European tick vector, Ixodes ricinus, encodes for defensin peptides with diverse antimicrobial activities against distantly related pathogens. These pathogens include fungi, Gram-negative and Gram-positive bacteria, i.e., a wide antimicrobial spectrum. Ticks do not transmit these pathogens, suggesting that these defensins may act against a wide range of microbes encountered by ticks during blood feeding or off-host periods. As demonstrated here, these I. ricinus defensins are also effective against the apicomplexan parasite Plasmodium falciparum. To study the general evolution of antimicrobial activity in tick defensins, the ancestral amino acid sequence of chelicerate defensins, which existed approximately 444 million years ago, was reconstructed using publicly available scorpion and tick defensin sequences (named Scorpions-Ticks Defensins Ancestor, STiDA. The activity of STiDA was tested against P. falciparum and the same Gram-negative and Gram-positive bacteria that were used for the I. ricinus defensins. While some extant tick defensins exhibit a wide antimicrobial spectrum, the ancestral defensin showed moderate activity against one of the tested microbes, P. falciparum. This study suggests that amino acid variability and defensin family expansion increased the antimicrobial spectrum of ancestral tick defensins.

Oil Palm Defensin: A Thermal Stable Peptide that Restricts the Mycelial Growth of Ganoderma boninense.

Science.gov (United States)

Tan, Yung-Chie; Ang, Cheng-Liang; Wong, Mui-Yun; Ho, Chai-Ling

2016-01-01

Plant defensins are plant defence peptides that have many different biological activities, including antifungal, antimicrobial, and insecticidal activities. A cDNA (EgDFS) encoding defensin was isolated from Elaeis guineensis. The open reading frame of EgDFS contained 231 nucleotides encoding a 71-amino acid protein with a predicted molecular weight at 8.69 kDa, and a potential signal peptide. The eight highly conserved cysteine sites in plant defensins were also conserved in EgDFS. The EgDFS sequence lacking 30 amino acid residues at its N-terminus (EgDFSm) was cloned into Escherichia coli BL21 (DE3) pLysS and successfully expressed as a soluble recombinant protein. The recombinant EgDFSm was found to be a thermal stable peptide which demonstrated inhibitory activity against the growth of G. boninense possibly by inhibiting starch assimilation. The role of EgDFSm in oil palm defence system against the infection of pathogen G. boninense was discussed.

Partial characterization of three β-defensin gene transcripts in river ...

African Journals Online (AJOL)

In this study, the tracheal tissues from Egyptian river buffalo and cattle were screened for the presence of three bovine β-defensin gene transcripts. Three primer pairs were designed on the basis of published Bos taurus sequences for partial amplification of β-defensin 4, β-defensin 10 and β-defensin 11 complementary DNA ...

A study on β-defensin-2 and histatin-5 as a diagnostic marker of early childhood caries progression

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Anna Jurczak

2015-01-01

Full Text Available BACKGROUND: Recently, a continuous growth of interest has been observed in antimicrobial peptides (AMPs in the light of an alarming increase in resistance of bacteria and fungi against antibiotics. AMPs are used as biomarkers in diagnosis and monitoring of oral cavity pathologies. Therefore, the determination of specific protein profiles in children diagnosed with early childhood caries (ECC might be a basis for effective screening tests and specialized examinations which may enable progression of disease METHODS: The objective of the studies was to determine the role of histatin-5 and β-defensing-2 as a diagnostic marker of early childhood caries progression. In this work, results of concentration determination of two salivary proteins (histatin-5 and β-defensin-2 were presented. In addition, bacterial profiles from dental plaque in various stages of ECC and control were marked. The assessment of alteration in the concentration of these two proteins in a study group of children with various stages of ECC and a control group consisting of children with no symptoms was performed by enzyme-linked immunosorbent assays RESULTS: The statistical analysis showed a significant increase in the concentration of histatin-5 and β-defensin-2 in the study group compared to the control group and correlated with the progression of the disease CONCLUSIONS: The confirmation of concentration changes in these proteins during the progression of dental caries may discover valuable disease progression biomarkers

ß-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria

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Joanna Baricelli

2015-02-01

Full Text Available OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA. Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n = 61 than in mature milk samples (n = 39, as follows: median and range, 8.52 (2.6-16.3 µg/ml versus 0.97 (0.22-3.78, p < 0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients.

The Alpha-defensin Test for Periprosthetic Joint Infections Is Not Affected by Prior Antibiotic Administration.

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Shahi, Alisina; Parvizi, Javad; Kazarian, Gregory S; Higuera, Carlos; Frangiamore, Salvatore; Bingham, Joshua; Beauchamp, Christopher; Valle, Craig Della; Deirmengian, Carl

2016-07-01

Previous studies have demonstrated that the administration of antibiotics to patients before performing diagnostic testing for periprosthetic joint infection (PJI) can interfere with the accuracy of test results. Although a single-institution study has suggested that alpha-defensin maintains its concentration and sensitivity even after antibiotic treatment, this has not yet been demonstrated in a larger multiinstitutional study. (1) For the evaluation of PJI, is prior antibiotic administration associated with decreased alpha-defensin levels? (2) When prior antibiotics are given, is alpha-defensin a better screening test for PJI than the traditional tests (erythrocyte sedimentation rate [ESR], C-reactive protein [CRP], fluid white blood cells, fluid polymorphonuclear cells [PMNs], and fluid culture)? This retrospective study included data from 106 hip and knee arthroplasties with Musculoskeletal Infection Society-defined PJI from four centers. Of the 106 patients in this study, 30 (28%) were treated with antibiotics for PJI before diagnostic workup (ABX group), and 76 (72%) were not treated before the diagnostic workup (NO-ABX group). There were no differences in age, sex, joint, culture-negative rate, or bacteriology between groups. The patients in the ABX group had antibiotics initiated by physicians who commenced care before assessment for PJI by the treating surgeon's service. We compared the alpha-defensin levels and sensitivity between the ABX and NO-ABX groups. Additionally, the sensitivity of the alpha-defensin test was compared to that of traditional tests for PJI among patients on antibiotics. The administration of antibiotics before performing the alpha-defensin test for PJI was not associated with a decreased median alpha-defensin level (ABX group, median 4.2 [range, 1.79-12.8 S/CO] versus NO-ABX, median 4.9 [range, 0.5-16.8 S/CO], difference of medians: 0.68 S/CO [95% confidence interval {CI}, -0.98 to 1.26], p = 0.451). Furthermore, the alpha-defensin

Novel aspects of defensins' involvement in virus-induced autoimmunity in the central nervous system.

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Kazakos, Evangelos I; Kountouras, Jannis; Polyzos, Stergios A; Deretzi, Georgia

2017-05-01

ultimately accumulates as protein deposits. It should be highlighted that β-amyloid brain deposits are a hallmark of many neurodegenerative diseases. During human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections, selective elicitation of the VH1-69 antibody subfamily directed against specific, viral glycoprotein epitopes, often correlates with a higher likelihood of developing autoimmune disorders. In addition, we propose that recently described defensin affinity towards the same epitopes may prevent proper viral fusion, but at the expense of augmenting autoimmune reactivity. Moreover, in the context of complex and largely unknown symbiotic relations between host microbiota and pathogenic viruses, we propose that co-stimulation of defensin expression may modulate adaptive immune responses thereby enhancing inflammatory cascades responsible for autoimmunity. Finally, we describe current evidence that supports the hypothesis of a possible involvement of defensins in HCV-related autoimmunity. Exploring further potential implications of human defensins in virus-related autoimmunity may lead to improved strategies for the treatment of neurodegenerative disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

α-Defensins and outcome in patients with chronic heart failure

DEFF Research Database (Denmark)

Christensen, Heidi M; Frystyk, Jan; Faber, Jens

2012-01-01

Aim a-Defensins are part of the innate immune system. Low-grade inflammation seems to play a crucial role in development and progression of chronic heart failure (CHF). The aims of the present study were to compare plasma levels of a-defensins in CHF patients and healthy controls and to examine......% confidence interval 1.19-2.28, P = 0.002) per 1 standard deviation increment in Ln (natural logarithm)-transformed a-defensin values. The combination of high a-defensins and NT-proBNP levels provided incremental prognostic information independent of well-known prognostic biomarkers in heart failure...... in 194 CHF patients, and compared plasma levels with those of 98 age-matched healthy controls. a-Defensin levels were twice as high among CHF patients in New York Heart Association (NYHA) functional class III-IV than in patients in NYHA class I-II and healthy controls (P = 0.001). The absolute increase...

Heterologous expression and solution structure of defensin from lentil Lens culinaris

International Nuclear Information System (INIS)

Shenkarev, Zakhar O.; Gizatullina, Albina K.; Finkina, Ekaterina I.; Alekseeva, Ekaterina A.; Balandin, Sergey V.; Mineev, Konstantin S.; Arseniev, Alexander S.; Ovchinnikova, Tatiana V.

2014-01-01

Highlights: • Lentil defensin Lc-def and its 15 N-labeled analog were overexpressed in E. coli. • Lc-def is active against fungi, but does not inhibit growth of G+ and G− bacteria. • Lc-def spatial structure involves triple-stranded β-sheet and α-helix (CSαβ motif). • Lc-def is able to bind to anionic lipid vesicles under low-salt conditions. • NMR data revealed significant μs–ms mobility in the loops 1 and 3 of Lc-def. - Abstract: A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4 Da and consists of 47 amino acid residues. Lc-def and its 15 N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs–ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt conditions. The Lc-def antifungal activity might be mediated through electrostatic interaction with anionic lipid components of fungal membranes

Heterologous expression and solution structure of defensin from lentil Lens culinaris

Energy Technology Data Exchange (ETDEWEB)

Shenkarev, Zakhar O. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, 117997 Moscow (Russian Federation); Gizatullina, Albina K. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700 Dolgoprudny, Moscow Region (Russian Federation); Finkina, Ekaterina I.; Alekseeva, Ekaterina A.; Balandin, Sergey V.; Mineev, Konstantin S. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, 117997 Moscow (Russian Federation); Arseniev, Alexander S. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700 Dolgoprudny, Moscow Region (Russian Federation); Ovchinnikova, Tatiana V., E-mail: ovch@ibch.ru [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700 Dolgoprudny, Moscow Region (Russian Federation)

2014-08-22

Highlights: • Lentil defensin Lc-def and its {sup 15}N-labeled analog were overexpressed in E. coli. • Lc-def is active against fungi, but does not inhibit growth of G+ and G− bacteria. • Lc-def spatial structure involves triple-stranded β-sheet and α-helix (CSαβ motif). • Lc-def is able to bind to anionic lipid vesicles under low-salt conditions. • NMR data revealed significant μs–ms mobility in the loops 1 and 3 of Lc-def. - Abstract: A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4 Da and consists of 47 amino acid residues. Lc-def and its {sup 15}N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs–ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt conditions. The Lc-def antifungal activity might be mediated through electrostatic interaction with anionic lipid components of fungal membranes.

C/EBPβ Promotes Immunity to Oral Candidiasis through Regulation of β-Defensins.

Science.gov (United States)

Simpson-Abelson, Michelle R; Childs, Erin E; Ferreira, M Carolina; Bishu, Shrinivas; Conti, Heather R; Gaffen, Sarah L

2015-01-01

Humans or mice subjected to immunosuppression, such as corticosteroids or anti-cytokine biologic therapies, are susceptible to mucosal infections by the commensal fungus Candida albicans. Recently it has become evident that the Th17/IL-17 axis is essential for immunity to candidiasis, but the downstream events that control immunity to this fungus are poorly understood. The CCAAT/Enhancer Binding Protein-β (C/EBPβ) transcription factor is important for signaling by multiple inflammatory stimuli, including IL-17. C/EBPβ is regulated in a variety of ways by IL-17, and controls several downstream IL-17 target genes. However, the role of C/EBPβ in vivo is poorly understood, in part because C/EBPβ-deficient mice are challenging to breed and work with. In this study, we sought to understand the role of C/EBPβ in the context of an IL-17-dependent immune response, using C. albicans infection as a model system. Confirming prior findings, we found that C/EBPβ is required for immunity to systemic candidiasis. In contrast, C/EBPβ(-/-) mice were resistant to oropharyngeal candidiasis (OPC), in a manner indistinguishable from immunocompetent WT mice. However, C/EBPβ(-/-) mice experienced more severe OPC than WT mice in the context of cortisone-induced immunosuppression. Expression of the antimicrobial peptide β-defensin (BD)-3 correlated strongly with susceptibility in C/EBPβ(-/-) mice, but no other IL-17-dependent genes were associated with susceptibility. Therefore, C/EBPβ contributes to immunity to mucosal candidiasis during cortisone immunosuppression in a manner linked to β-defensin 3 expression, but is apparently dispensable for the IL-17-dependent response.

Anti-Legionella dumoffii Activity of Galleria mellonella Defensin and Apolipophorin III

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Małgorzata Cytryńska

2012-12-01

Full Text Available The gram-negative bacterium Legionella dumoffii is, beside Legionella pneumophila, an etiological agent of Legionnaires’ disease, an atypical form of pneumonia. The aim of this study was to determine the antimicrobial activity of Galleria mellonella defense polypeptides against L. dumoffii. The extract of immune hemolymph, containing a mixture of defense peptides and proteins, exhibited a dose-dependent bactericidal effect on L. dumoffii. The bacterium appeared sensitive to a main component of the hemolymph extract, apolipophorin III, as well as to a defense peptide, Galleria defensin, used at the concentrations 0.4 mg/mL and 40 μg/mL, respectively. L. dumoffii cells cultured in the presence of choline were more susceptible to both defense factors analyzed. A transmission electron microscopy study of bacterial cells demonstrated that Galleria defensin and apolipophorin III induced irreversible cell wall damage and strong intracellular alterations, i.e., increased vacuolization, cytoplasm condensation and the appearance of electron-white spaces in electron micrographs. Our findings suggest that insects, such as G. mellonella, with their great diversity of antimicrobial factors, can serve as a rich source of compounds for the testing of Legionella susceptibility to defense-related peptides and proteins.

Vitamin D Signaling Through Induction of Paneth Cell Defensins Maintains Gut Microbiota and Improves Metabolic Disorders and Hepatic Steatosis in Animal Models

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Danmei Su

2016-11-01

Full Text Available Metabolic syndrome (MetS, characterized as obesity, insulin resistance, and non-alcoholic fatty liver diseases (NAFLD，is associated with vitamin D insufficiency/deficiency in epidemiological studies, while the underlying mechanism is poorly addressed. On the other hand, disorder of gut microbiota, namely dysbiosis, is known to cause MetS and NAFLD. It is also known that systemic inflammation blocks insulin signaling pathways, leading to insulin resistance and glucose intolerance, which are the driving force for hepatic steatosis. Vitamin D receptor (VDR is highly expressed in the ileum of the small intestine，which prompted us to test a hypothesis that vitamin D signaling may determine the enterotype of gut microbiota through regulating the intestinal interface. Here, we demonstrate that high-fat-diet feeding (HFD is necessary but not sufficient, while additional vitamin D deficiency (VDD as a second hit is needed, to induce robust insulin resistance and fatty liver. Under the two hits (HFD+VDD, the Paneth cell-specific alpha-defensins including α-defensin 5 (DEFA5, MMP7 which activates the pro-defensins, as well as tight junction genes, and MUC2 are all suppressed in the ileum, resulting in mucosal collapse, increased gut permeability, dysbiosis, endotoxemia, systemic inflammation which underlie insulin resistance and hepatic steatosis. Moreover, under the vitamin D deficient high fat feeding (HFD+VDD, Helicobacter hepaticus, a known murine hepatic-pathogen, is substantially amplified in the ileum, while Akkermansia muciniphila, a beneficial symbiotic, is diminished. Likewise, the VD receptor (VDR knockout mice exhibit similar phenotypes, showing down regulation of alpha-defensins and MMP7 in the ileum, increased Helicobacter hepaticus and suppressed Akkermansia muciniphila. Remarkably, oral administration of DEFA5 restored eubiosys, showing suppression of Helicobacter hepaticus and increase of Akkermansia muciniphila in association with

The Fungal Defensin Family Enlarged

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Jiajia Wu

2014-08-01

Full Text Available Fungi are an emerging source of peptide antibiotics. With the availability of a large number of model fungal genome sequences, we can expect that more and more fungal defensin-like peptides (fDLPs will be discovered by sequence similarity search. Here, we report a total of 69 new fDLPs encoded by 63 genes, in which a group of fDLPs derived from dermatophytes are defined as a new family (fDEF8 according to sequence and phylogenetic analyses. In the oleaginous fungus Mortierella alpine, fDLPs have undergone extensive gene expansion. Our work further enlarges the fungal defensin family and will help characterize new peptide antibiotics with therapeutic potential.

Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis.

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Telleria, Erich L; Sant'Anna, Maurício R Viana; Alkurbi, Mohammad O; Pitaluga, André N; Dillon, Rod J; Traub-Csekö, Yara M

2013-01-11

Phlebotomine insects harbor bacterial, viral and parasitic pathogens that can cause diseases of public health importance. Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the New World. Insects can mount a powerful innate immune response to pathogens. Defensin peptides take part in this response and are known to be active against Gram-positive and Gram-negative bacteria, and some parasites. We studied the expression of a defensin gene from Lutzomyia longipalpis to understand its role in sand fly immune response. We identified, sequenced and evaluated the expression of a L. longipalpis defensin gene by semi-quantitative RT-PCR. The gene sequence was compared to other vectors defensins and expression was determined along developmental stages and after exposure of adult female L. longipalpis to bacteria and Leishmania. Phylogenetic analysis showed that the L. longipalpis defensin is closely related to a defensin from the Old World sand fly Phlebotomus duboscqi. Expression was high in late L4 larvae and pupae in comparison to early larval stages and newly emerged flies. Defensin expression was modulated by oral infection with bacteria. The Gram-positive Micrococcus luteus induced early high defensin expression, whilst the Gram-negative entomopathogenic Serratia marcescens induced a later response. Bacterial injection also induced defensin expression in adult insects. Female sand flies infected orally with Leishmania mexicana showed no significant difference in defensin expression compared to blood fed insects apart from a lower defensin expression 5 days post Leishmania infection. When Leishmania was introduced into the hemolymph by injection there was no induction of defensin expression until 72 h later. Our results suggest that L. longipalpis modulates defensin expression upon bacterial and Leishmania infection, with patterns of expression that are distinct among bacterial species and routes of infection.

Spatio-Temporal Expression Patterns of Arabidopsis thaliana and Medicago truncatula Defensin-Like Genes

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Nallu, Sumitha; Wang, Lin; Botanga, Christopher J.; Gomez, S. Karen; Costa, Liliana M.; Harrison, Maria J.; Samac, Deborah A.; Glazebrook, Jane; Katagiri, Fumiaki; Gutierrez-Marcos, Jose F.; VandenBosch, Kathryn A.

2013-01-01

Plant genomes contain several hundred defensin-like (DEFL) genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species. PMID:23527067

Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes.

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Mesfin Tesfaye

Full Text Available Plant genomes contain several hundred defensin-like (DEFL genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species.

Rabbit defensin (NP-1) genetic engineering of plant | Ting | African ...

African Journals Online (AJOL)

Rabbit defensin (NP-1) genetic engineering of plant. ... Log in or Register to get access to full text downloads. ... defensin genetic engineering of plant in recent years, and also focuses on the existing problems and new strategies in this area.

Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.

Science.gov (United States)

Gerashchenko, O L; Zhuravel, E V; Skachkova, O V; Khranovska, N N; Filonenko, V V; Pogrebnoy, P V; Soldatkina, M A

2013-06-01

The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.

Enteric alpha defensins in norm and pathology

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Lisitsyn Nikolai A

2012-01-01

Full Text Available Abstract Microbes living in the mammalian gut exist in constant contact with immunity system that prevents infection and maintains homeostasis. Enteric alpha defensins play an important role in regulation of bacterial colonization of the gut, as well as in activation of pro- and anti-inflammatory responses of the adaptive immune system cells in lamina propria. This review summarizes currently available data on functions of mammalian enteric alpha defensins in the immune defense and changes in their secretion in intestinal inflammatory diseases and cancer.

Directional and balancing selection in human beta-defensins.

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Hollox, Edward J; Armour, John A L

2008-04-16

In primates, infection is an important force driving gene evolution, and this is reflected in the importance of infectious disease in human morbidity today. The beta-defensins are key components of the innate immune system, with antimicrobial and cell signalling roles, but also reproductive functions. Here we examine evolution of beta-defensins in catarrhine primates and variation within different human populations. We show that five beta-defensin genes that do not show copy number variation in humans show evidence of positive selection in catarrhine primates, and identify specific codons that have been under selective pressure. Direct haplotyping of DEFB127 in humans suggests long-term balancing selection: there are two highly diverged haplotype clades carrying different variants of a codon that, in primates, is positively selected. For DEFB132, we show that extensive diversity, including a four-state amino acid polymorphism (valine, isoleucine, alanine and threonine at position 93), is present in hunter-gatherer populations, both African and non-African, but not found in samples from agricultural populations. Some, but not all, beta-defensin genes show positive selection in catarrhine primates. There is suggestive evidence of different selective pressures on these genes in humans, but the nature of the selective pressure remains unclear and is likely to differ between populations.

Inflammatory disorders mimicking periprosthetic joint infections may result in false positive α-defensin.

Science.gov (United States)

Plate, Andreas; Stadler, Laura; Sutter, Reto; Anagnostopoulos, Alexia; Frustaci, Dario; Zbinden, Reinhard; Fucentese, Sandro F; Zinkernagel, Annelies S; Zingg, Patrick O; Achermann, Yvonne

2018-02-26

The antimicrobial peptide α-defensin has recently been introduced as potential "single" biomarker with a high sensitivity and specificity for the preoperative diagnosis of periprosthetic joint infections (PJIs). However, most studies assessed the benefits of the test with exclusion of patients with rheumatic diseases. We aimed to evaluate the α-defensin test in a cohort study without exclusion of cases with inflammatory diseases. Between June 2016 and June 2017, we prospectively included cases with a suspected PJI and an available lateral flow test α-defensin (Synovasure®) in synovial fluid. We compared the test result to the diagnostic criteria for PJIs published by an International Consensus Group in 2013. We included 109 cases (49 hips, 60 knees) in which preoperative α-defensin tests had been performed. Thereof, 20 PJIs (16 hips, 4 knees) were diagnosed. Preoperative α-defensin tests were positive in 25 cases (22.9%) with a test sensitivity and specificity of 90% and 92.1% (95% confidence interval [CI], 68.3 - 98.8% and 84.5 - 96.8%, respectively), and a high negative predictive value of 97.6% (95% CI, 91.7 - 99.4%). We interpreted seven α-defensin tests as false positive, mainly in cases with inflammatory rheumatic diseases, including crystal deposition diseases. A negative synovial α-defensin test can reliably rule out a PJI. However, the test can be false positive in conjunction with an underlying non-infectious inflammatory disease. We therefore propose to use the α-defensin test only in addition to MSIS criteria and assessment for crystals in synovial aspirates. Copyright © 2018. Published by Elsevier Ltd.

Directional and balancing selection in human beta-defensins

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Armour John AL

2008-04-01

Full Text Available Abstract Background In primates, infection is an important force driving gene evolution, and this is reflected in the importance of infectious disease in human morbidity today. The beta-defensins are key components of the innate immune system, with antimicrobial and cell signalling roles, but also reproductive functions. Here we examine evolution of beta-defensins in catarrhine primates and variation within different human populations. Results We show that five beta-defensin genes that do not show copy number variation in humans show evidence of positive selection in catarrhine primates, and identify specific codons that have been under selective pressure. Direct haplotyping of DEFB127 in humans suggests long-term balancing selection: there are two highly diverged haplotype clades carrying different variants of a codon that, in primates, is positively selected. For DEFB132, we show that extensive diversity, including a four-state amino acid polymorphism (valine, isoleucine, alanine and threonine at position 93, is present in hunter-gatherer populations, both African and non-African, but not found in samples from agricultural populations. Conclusion Some, but not all, beta-defensin genes show positive selection in catarrhine primates. There is suggestive evidence of different selective pressures on these genes in humans, but the nature of the selective pressure remains unclear and is likely to differ between populations.

Purification, cDNA cloning and modification of a defensin from the coconut rhinoceros beetle, Oryctes rhinoceros.

Science.gov (United States)

Ishibashi, J; Saido-Sakanaka, H; Yang, J; Sagisaka, A; Yamakawa, M

1999-12-01

A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros, immunized with Escherichia coli. A full-size cDNA was cloned by combining reverse-transcription PCR (RT-PCR), and 5'- and 3'-rapid amplification of cDNA ends (RACE). Analysis of the O. rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpighian tubules. O. rhinoceros defensin showed strong antibacterial activity against Staphylococcus aureus. A 9-mer peptide amidated at its C-terminus, AHCLAICRK-NH2 (Ala22-Lys30-NH2), was synthesized based on the deduced amino-acid sequence, assumed to be an active site sequence by analogy with the sequence of a defensin isolated from larvae of the beetle Allomyrina dichotoma. This peptide showed antibacterial activity against S. aureus, methicillin-resistant S. aureus, E. coli and Pseudomonas aeruginosa. We further modified this oligopeptide and synthesized five 9-mer peptides, ALRLAIRKR-NH2, ALLLAIRKR-NH2, AWLLAIRKR-NH2, ALYLAIRKR-NH2 and ALWLAIRKR-NH2. These oligopeptides showed strong antibacterial activity against Gram-negative and Gram-positive bacteria. The antibacterial effect of Ala22-Lys30-NH2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. These Ala22-Lys30-NH2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast cells or macrophages, except for AWLLAIRKR-NH2.

Contribution of alpha- and beta-defensins to lung function decline and infection in smokers: an association study

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Anthonisen Nicholas R

2006-05-01

Full Text Available Abstract Background Alpha-defensins, which are major constituents of neutrophil azurophilic granules, and beta-defensins, which are expressed in airway epithelial cells, could contribute to the pathogenesis of chronic obstructive pulmonary disease by amplifying cigarette smoke-induced and infection-induced inflammatory reactions leading to lung injury. In Japanese and Chinese populations, two different beta-defensin-1 polymorphisms have been associated with chronic obstructive pulmonary disease phenotypes. We conducted population-based association studies to test whether alpha-defensin and beta-defensin polymorphisms influenced smokers' susceptibility to lung function decline and susceptibility to lower respiratory infection in two groups of white participants in the Lung Health Study (275 = fast decline in lung function and 304 = no decline in lung function. Methods Subjects were genotyped for the alpha-defensin-1/alpha-defensin-3 copy number polymorphism and four beta-defensin-1 polymorphisms (G-20A, C-44G, G-52A and Val38Ile. Results There were no associations between individual polymorphisms or imputed haplotypes and rate of decline in lung function or susceptibility to infection. Conclusion These findings suggest that, in a white population, the defensin polymorphisms tested may not be of importance in determining who develops abnormally rapid lung function decline or is susceptible to developing lower respiratory infections.

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

NARCIS (Netherlands)

Snijder, Joost; Van De Waterbeemd, Michiel; Glover, Matthew S.; Shi, Liuqing; Clemmer, David E.; Heck, Albert J R

2015-01-01

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions.

DIRProt: a computational approach for discriminating insecticide resistant proteins from non-resistant proteins.

Science.gov (United States)

Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Banchariya, Anjali; Rao, Atmakuri Ramakrishna

2017-03-24

Insecticide resistance is a major challenge for the control program of insect pests in the fields of crop protection, human and animal health etc. Resistance to different insecticides is conferred by the proteins encoded from certain class of genes of the insects. To distinguish the insecticide resistant proteins from non-resistant proteins, no computational tool is available till date. Thus, development of such a computational tool will be helpful in predicting the insecticide resistant proteins, which can be targeted for developing appropriate insecticides. Five different sets of feature viz., amino acid composition (AAC), di-peptide composition (DPC), pseudo amino acid composition (PAAC), composition-transition-distribution (CTD) and auto-correlation function (ACF) were used to map the protein sequences into numeric feature vectors. The encoded numeric vectors were then used as input in support vector machine (SVM) for classification of insecticide resistant and non-resistant proteins. Higher accuracies were obtained under RBF kernel than that of other kernels. Further, accuracies were observed to be higher for DPC feature set as compared to others. The proposed approach achieved an overall accuracy of >90% in discriminating resistant from non-resistant proteins. Further, the two classes of resistant proteins i.e., detoxification-based and target-based were discriminated from non-resistant proteins with >95% accuracy. Besides, >95% accuracy was also observed for discrimination of proteins involved in detoxification- and target-based resistance mechanisms. The proposed approach not only outperformed Blastp, PSI-Blast and Delta-Blast algorithms, but also achieved >92% accuracy while assessed using an independent dataset of 75 insecticide resistant proteins. This paper presents the first computational approach for discriminating the insecticide resistant proteins from non-resistant proteins. Based on the proposed approach, an online prediction server DIRProt has

Plasmid-mediated resistance to thrombin-induced platelet microbicidal protein in staphylococci: role of the qacA locus.

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Kupferwasser, L I; Skurray, R A; Brown, M H; Firth, N; Yeaman, M R; Bayer, A S

1999-10-01

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded staphylococcal gene qacA mediates multidrug resistance to multiple organic cations via a proton motive force-dependent efflux pump. We studied whether the qacA gene might also confer resistance to cationic tPMP-1. Staphylococcal plasmids encoding qacA were found to confer resistance to tPMP-1 in an otherwise susceptible parental strain. Deletions which removed the region containing the qacA gene in the S. aureus multiresistance plasmid pSK1 abolished tPMP-1 resistance. Resistance to tPMP-1 in the qacA-bearing strains was inoculum independent but peptide concentration dependent, with the level of resistance decreasing at higher peptide concentrations for a given inoculum. There was no apparent cross-resistance in qacA-bearing strains to other endogenous cationic antimicrobial peptides which are structurally distinct from tPMP-1, including human neutrophil defensin 1, protamine, or the staphylococcal lantibiotics pep5 and nisin. These data demonstrate that the staphylococcal multidrug resistance gene qacA also mediates in vitro resistance to cationic tPMP-1.

Antibacterial and antiviral roles of a fish β-defensin expressed both in pituitary and testis.

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Jun-Yan Jin

Full Text Available Defensins are a group of cationic peptides that exhibit broad-spectrum antimicrobial activity. In this study, we cloned and characterized a β-defensin from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides. Interestingly, the β-defensin was shown to be dominantly expressed in pituitary and testis by RT-PCR and Western blot analysis, and its transcript level is significantly upregulated in reproduction organs from intersexual gonad to testis during the natural and artificial sex reversal. Promoter sequence and the responsible activity region analyses revealed the pituitary-specific POU1F1a transcription binding site and testis-specific SRY responsible site, and demonstrated that the pituitary-specific POU1F1a transcription binding site that locates between -180 and -208 bp is the major responsible region of grouper β-defensin promoter activity. Immunofluorescence localization observed its pituicyte expression in pituitary and spermatogonic cell expression in testis. Moreover, both in vitro antibacterial activity assay of the recombinant β-defensin and in vivo embryo microinjection of the β-defensin mRNA were shown to be effective in killing gram-negative bacteria. And, its antiviral role was also demonstrated in EPC cells transfected with the β-defensin construct. Additionally, the antibacterial activity was sensitive to concentrations of Na(+, K(+, Ca(2+ and Mg(2+. The above intriguing findings strongly suggest that the fish β-defensin might play significant roles in both innate immunity defense and reproduction endocrine regulation.

Antibacterial and antiviral roles of a fish β-defensin expressed both in pituitary and testis.

Science.gov (United States)

Jin, Jun-Yan; Zhou, Li; Wang, Yang; Li, Zhi; Zhao, Jiu-Gang; Zhang, Qi-Ya; Gui, Jian-Fang

2010-12-20

Defensins are a group of cationic peptides that exhibit broad-spectrum antimicrobial activity. In this study, we cloned and characterized a β-defensin from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides). Interestingly, the β-defensin was shown to be dominantly expressed in pituitary and testis by RT-PCR and Western blot analysis, and its transcript level is significantly upregulated in reproduction organs from intersexual gonad to testis during the natural and artificial sex reversal. Promoter sequence and the responsible activity region analyses revealed the pituitary-specific POU1F1a transcription binding site and testis-specific SRY responsible site, and demonstrated that the pituitary-specific POU1F1a transcription binding site that locates between -180 and -208 bp is the major responsible region of grouper β-defensin promoter activity. Immunofluorescence localization observed its pituicyte expression in pituitary and spermatogonic cell expression in testis. Moreover, both in vitro antibacterial activity assay of the recombinant β-defensin and in vivo embryo microinjection of the β-defensin mRNA were shown to be effective in killing gram-negative bacteria. And, its antiviral role was also demonstrated in EPC cells transfected with the β-defensin construct. Additionally, the antibacterial activity was sensitive to concentrations of Na(+), K(+), Ca(2+) and Mg(2+). The above intriguing findings strongly suggest that the fish β-defensin might play significant roles in both innate immunity defense and reproduction endocrine regulation.

Genome-Wide Sensitivity Analysis of the Microsymbiont Sinorhizobium meliloti to Symbiotically Important, Defensin-Like Host Peptides

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Markus F. F. Arnold

2017-08-01

Full Text Available The model legume species Medicago truncatula expresses more than 700 nodule-specific cysteine-rich (NCR signaling peptides that mediate the differentiation of Sinorhizobium meliloti bacteria into nitrogen-fixing bacteroids. NCR peptides are essential for a successful symbiosis in legume plants of the inverted-repeat-lacking clade (IRLC and show similarity to mammalian defensins. In addition to signaling functions, many NCR peptides exhibit antimicrobial activity in vitro and in vivo. Bacterial resistance to these antimicrobial activities is likely to be important for symbiosis. However, the mechanisms used by S. meliloti to resist antimicrobial activity of plant peptides are poorly understood. To address this, we applied a global genetic approach using transposon mutagenesis followed by high-throughput sequencing (Tn-seq to identify S. meliloti genes and pathways that increase or decrease bacterial competitiveness during exposure to the well-studied cationic NCR247 peptide and also to the unrelated model antimicrobial peptide polymyxin B. We identified 78 genes and several diverse pathways whose interruption alters S. meliloti resistance to NCR247. These genes encode the following: (i cell envelope polysaccharide biosynthesis and modification proteins, (ii inner and outer membrane proteins, (iii peptidoglycan (PG effector proteins, and (iv non-membrane-associated factors such as transcriptional regulators and ribosome-associated factors. We describe a previously uncharacterized yet highly conserved peptidase, which protects S. meliloti from NCR247 and increases competitiveness during symbiosis. Additionally, we highlight a considerable number of uncharacterized genes that provide the basis for future studies to investigate the molecular basis of symbiotic development as well as chronic pathogenic interactions.

Plasma alpha-defensin is associated with cardiovascular morbidity and mortality in type 1 diabetic patients

DEFF Research Database (Denmark)

Joseph, G.; Tarnow, L.; Astrup, A.S.

2008-01-01

CONTEXT: alpha-Defensins are antimicrobial peptides of the innate immune system. In addition, experimental evidence suggests that alpha-defensins are proatherogenic. OBJECTIVE: The objective of the study was to examine the predictive value of plasma alpha-defensin as a clinical marker of cardiova...... to the development of CVD or an innocent bystander Udgivelsesdato: 2008/4...

α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

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Mayim E. Wiens

2017-01-01

Full Text Available α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5 blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses.

Characterization of the antimicrobial peptide family defensins in the Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus), and tammar wallaby (Macropus eugenii).

Science.gov (United States)

Jones, Elizabeth A; Cheng, Yuanyuan; O'Meally, Denis; Belov, Katherine

2017-03-01

Defensins comprise a family of cysteine-rich antimicrobial peptides with important roles in innate and adaptive immune defense in vertebrates. We characterized alpha and beta defensin genes in three Australian marsupials: the Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus), and tammar wallaby (Macropus eugenii) and identified 48, 34, and 39 defensins, respectively. One hundred and twelve have the classical antimicrobial peptides characteristics required for pathogen membrane targeting, including cationic charge (between 1+ and 15+) and a high proportion of hydrophobic residues (>30%). Phylogenetic analysis shows that gene duplication has driven unique and species-specific expansions of devil, koala, and tammar wallaby beta defensins and devil alpha defensins. Defensin genes are arranged in three genomic clusters in marsupials, whereas further duplications and translocations have occurred in eutherians resulting in four and five gene clusters in mice and humans, respectively. Marsupial defensins are generally under purifying selection, particularly residues essential for defensin structural stability. Certain hydrophobic or positively charged sites, predominantly found in the defensin loop, are positively selected, which may have functional significance in defensin-target interaction and membrane insertion.

Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

Science.gov (United States)

Guillén-Chable, Francisco; Arenas-Sosa, Iván; Islas-Flores, Ignacio; Corzo, Gerardo; Martinez-Liu, Cynthia; Estrada, Georgina

2017-08-01

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa. Copyright © 2017 Elsevier Inc. All rights reserved.

Interaction of Defensins with Model Cell Membranes

Science.gov (United States)

Sanders, Lori K.; Schmidt, Nathan W.; Yang, Lihua; Mishra, Abhijit; Gordon, Vernita D.; Selsted, Michael E.; Wong, Gerard C. L.

2009-03-01

Antimicrobial peptides (AMPs) comprise a key component of innate immunity for a wide range of multicellular organisms. For many AMPs, activity comes from their ability to selectively disrupt and lyse bacterial cell membranes. There are a number of proposed models for this action, but the detailed molecular mechanism of selective membrane permeation remains unclear. Theta defensins are circularized peptides with a high degree of selectivity. We investigate the interaction of model bacterial and eukaryotic cell membranes with theta defensins RTD-1, BTD-7, and compare them to protegrin PG-1, a prototypical AMP, using synchrotron small angle x-ray scattering (SAXS). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane phase behavior induced by these different peptides we will discuss the importance of amino acid composition and placement on membrane rearrangement.

Identification of structural traits that increase the antimicrobial activity of a chimeric peptide of human β-defensins 2 and 3.

Science.gov (United States)

Spudy, Björn; Sönnichsen, Frank D; Waetzig, Georg H; Grötzinger, Joachim; Jung, Sascha

2012-10-12

Antimicrobial peptides participate in the first line of defence of many organisms against pathogens. In humans, the family of β-defensins plays a pivotal role in innate immunity. Two human β-defensins, β-defensin-2 and -3 (HBD2 and HBD3), show substantial sequence identity and structural similarity. However, HBD3 kills Staphylococcus (S.) aureus with a 4- to 8-fold higher efficiency compared to HBD2, whereas their activities against Escherichia (E.) coli are very similar. The generation of six HBD2/HBD3-chimeric molecules led to the identification of distinct molecular regions which mediate their divergent killing properties. One of the chimeras (chimera C3) killed both E. coli and S. aureus with an even higher efficacy compared to the wild-type molecules. Due to the broad spectrum of its antimicrobial activity against many human multidrug-resistant pathogens, this HBD2/HBD3-chimeric peptide represents a promising candidate for a new class of antibiotics. In order to investigate the structural basis of its exceptional antimicrobial activity, the peptide's tertiary structure was determined by NMR spectroscopy, which allowed its direct comparison to the published structures of HBD2 and HBD3 and the identification of the activity-increasing molecular features. Copyright © 2012 Elsevier Inc. All rights reserved.

High level expression of human epithelial β-defensins (hBD-1, 2 and 3 in papillomavirus induced lesions

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Chong Kong T

2006-09-01

Full Text Available Abstract Background Epithelial defensins including human β-defensins (hBDs and α-defensins (HDs are antimicrobial peptides that play important roles in the mucosal defense system. However, the role of defensins in papillomavirus induced epithelial lesions is unknown. Results Papilloma tissues were prospectively collected from 15 patients with recurrent respiratory papillomatosis (RRP and analyzed for defensins and chemokine IL-8 expression by quantitative, reverse-transcriptase polymerase chain reaction (RT-PCR assays. HBD-1, -2 and -3 mRNAs were detectable in papilloma samples from all RRP patients and the levels were higher than in normal oral mucosal tissues from healthy individuals. Immunohistochemical analysis showed that both hBD-1 and 2 were localized in the upper epithelial layers of papilloma tissues. Expression of hBD-2 and hBD-3 appeared to be correlated as indicated by scatter plot analysis (r = 0.837, p Conclusion Human β-defensins are upregulated in respiratory papillomas. This novel finding suggests that hBDs might contribute to innate and adaptive immune responses targeted against papillomavirus-induced epithelial lesions.

Expression and antimicrobial function of beta-defensin 1 in the lower urinary tract.

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Brian Becknell

Full Text Available Beta defensins (BDs are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1 expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1(-/- mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1(-/- and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract.

Expression and Antimicrobial Function of Beta-Defensin 1 in the Lower Urinary Tract

Science.gov (United States)

Becknell, Brian; Spencer, John David; Carpenter, Ashley R.; Chen, Xi; Singh, Aspinder; Ploeger, Suzanne; Kline, Jennifer; Ellsworth, Patrick; Li, Birong; Proksch, Ehrhardt; Schwaderer, Andrew L.; Hains, David S.; Justice, Sheryl S.; McHugh, Kirk M.

2013-01-01

Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1 -/-) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1 -/- and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract. PMID:24204930

Molecular Dynamics Simulations Reveal the Conformational Flexibility of Lipid II and Its Loose Association with the Defensin Plectasin in the Staphylococcus aureus Membrane

DEFF Research Database (Denmark)

Witzke, Sarah; Petersen, Michael; Carpenter, Timothy S.

2016-01-01

dynamics simulation study of the conformational dynamics of Lipid II within a detailed model of the Staphylococcus aureus cell membrane. We show that Lipid II is able to adopt a range of conformations, even within the packed lipidic environment of the membrane. Our simulations also reveal dimerization...... the biosynthesis of the cell wall. Given the urgent need for development of novel antibiotics to counter the growing threat of bacterial infection resistance, it is imperative that a thorough molecular-level characterization of the molecules targeted by antibiotics be achieved. To this end, we present a molecular...... of Lipid II mediated by cations. In the presence of the defensin peptide plectasin, the conformational lability of Lipid II allows it to form loose complexes with the protein, via a number of different binding modes....

Rabbit defensin (NP-1) genetic engineering of plant

African Journals Online (AJOL)

Jane

2011-08-22

Aug 22, 2011 ... rabbit defensin has a significant toxic effect on mouse tumor cells .... Disease is one of the important factors which lead to decrease of .... Transgenic Nitrate Reductase Deficient Mutant of Chlorella ellipsoide. J. Agric.

Alpha-defensin DEFA1A3 gene copy number elevation in Danish Crohn's disease patients

DEFF Research Database (Denmark)

Jespersgaard, Cathrine; Fode, Peder; Dybdahl, Marianne

2011-01-01

BACKGROUND AND PURPOSE OF STUDY: Extensive copy number variation is observed for the DEFA1A3 gene encoding alpha-defensins 1-3. The objective of this study was to determine the involvement of alpha-defensins in colonic tissue from Crohn's disease (CD) patients and the possible genetic association...... of DEFA1A3 with CD. METHODS: Two-hundred and forty ethnic Danish CD patients were included in the study. Reverse transcriptase PCR assays determined DEFA1A3 expression in colonic tissue from a subset of patients. Immunohistochemical analysis identified alpha-defensin peptides in colonic tissue. Copy...

Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

Energy Technology Data Exchange (ETDEWEB)

Gao, Bin [Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, 100101 Beijing (China); Zhu, Shunyi, E-mail: Zhusy@ioz.ac.cn [Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, 100101 Beijing (China)

2012-10-05

Highlights: Black-Right-Pointing-Pointer Al-M is an engineered fungal defensin with the n-loop of an insect defensin. Black-Right-Pointing-Pointer Al-M adopts a native defensin-like structure with high antibacterial potency. Black-Right-Pointing-Pointer Al-M kills bacteria through a membrane disruptive mechanism. Black-Right-Pointing-Pointer This work sheds light on the functional evolution of CS{alpha}{beta}-type defensins. -- Abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized {alpha}-helical and {beta}-sheet (CS{alpha}{beta}) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76 {mu}M. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

International Nuclear Information System (INIS)

Gao, Bin; Zhu, Shunyi

2012-01-01

Highlights: ► Al-M is an engineered fungal defensin with the n-loop of an insect defensin. ► Al-M adopts a native defensin-like structure with high antibacterial potency. ► Al-M kills bacteria through a membrane disruptive mechanism. ► This work sheds light on the functional evolution of CSαβ-type defensins. -- Abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized α-helical and β-sheet (CSαβ) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76 μM. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

The Arabidopsis mutant iop1 exhibits induced over-expression of the plant defensin gene PDF1.2 and enhanced pathogen resistance

NARCIS (Netherlands)

Penninckx, I.A.M.A.; Eggermont, K.; Schenk, P.M.; Ackerveken, van den G.; Cammue, B.P.A.; Thomma, B.P.H.J.

2003-01-01

Jasmonate and ethylene are concomitantly involved in the induction of the Arabidopsis plant defensin gene PDF1.2. To define genes in the signal transduction pathway leading to the induction of PDF1.2, we screened for mutants with induced over-expression of a β-glucuronidase reporter, under the

Structural and functional characterization of the conserved salt bridge in mammalian paneth cell alpha-defensins

DEFF Research Database (Denmark)

Rosengren, K Johan; Daly, Norelle L; Fornander, Liselotte M

2006-01-01

alpha-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and a mutant (E15D)-C...

Beta-Defensin-2 and Beta-Defensin-3 Reduce Intestinal Damage Caused by Salmonella typhimurium Modulating the Expression of Cytokines and Enhancing the Probiotic Activity of Enterococcus faecium

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Alessandra Fusco

2017-01-01

Full Text Available The intestinal microbiota is a major factor in human health and disease. This microbial community includes autochthonous (permanent inhabitants and allochthonous (transient inhabitants microorganisms that contribute to maintaining the integrity of the intestinal wall, modulating responses to pathogenic noxae and representing a key factor in the maturation of the immune system. If this healthy microbiota is disrupted by antibiotics, chemotherapy, or a change in diet, intestinal colonization by pathogenic bacteria or viruses may occur, leading to disease. To manage substantial microbial exposure, epithelial surfaces of the intestinal tract produce a diverse arsenal of antimicrobial peptides (AMPs, including, of considerable importance, the β-defensins, which directly kill or inhibit the growth of microorganisms. Based on the literature data, the purpose of this work was to create a line of intestinal epithelial cells able to stably express gene encoding human β-defensin-2 (hBD-2 and human β-defensin-3 (hBD-3, in order to test their role in S. typhimurium infections and their interaction with the bacteria of the gut microbiota.

Transgenic approaches for development of disease resistance in banana

International Nuclear Information System (INIS)

Shekhawat, Upendra K.S.; Ghag, Siddhesh B.; Ganapathi, Thumballi R.

2014-01-01

Banana (Musa spp.) is an important food and cash crop worldwide. Diseases and pests pose the most serious constraint to banana cultivation. Among the diseases, Fusarium wilt and Banana Bunchy Top Virus (BBTV) are the most important economically. We have explored different transgenic approaches for development of efficient resistance in banana against these two diseases. For countering Fusarium wilt, we have over expressed Petunia floral defensins using a strong constitutive promoter in transgenic banana plants. We have also tested a host induced gene silencing strategy targeting two vital fungal genes to obtain Fusarium resistant banana plants. For development of BBTV resistant banana plants also, we have used a host-induced gene silencing approach utilizing the full and partial coding sequence of the viral replication initiation protein. Successful bioassays performed in controlled greenhouse conditions have shown the efficacy of using these strategies to develop disease resistant banana plants. (author)

A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion.

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Matthew P Wood

Full Text Available Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.

Differential Susceptibility of Bacteria to Mouse Paneth Cell a-Defensins under Anaerobic Conditions

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Jennifer R. Mastroianni

2014-10-01

Full Text Available Small intestinal Paneth cells secrete a-defensin peptides, termed cryptdins (Crps in mice, into the intestinal lumen, where they confer immunity to oral infections and define the composition of the ileal microbiota. In these studies, facultative bacteria maintained under aerobic or anaerobic conditions displayed differential sensitivities to mouse a-defensins under in vitro assay conditions. Regardless of oxygenation, Crps 2 and 3 had robust and similar bactericidal activities against S. typhimurium and S. flexneri, but Crp4 activity against S. flexneri was attenuated in the absence of oxygen. Anaerobic bacteria varied in their susceptibility to Crps 2-4, with Crp4 showing less activity than Crps 2 and 3 against Enterococcus faecalis, and Bacteroides fragilis in anaerobic assays, but Fusobacterium necrophorum was killed only by Crp4 and not by Crps 2 and 3. The influence of anaerobiosis in modulating Crp bactericidal activities in vitro suggests that a-defensin effects on the enteric microbiota may be subject to regulation by local oxygen tension.

β-Defensin genomic copy number does not influence the age of onset in Huntington's Disease.

Science.gov (United States)

Vittori, Angelica; Orth, Michael; Roos, Raymund A C; Outeiro, Tiago F; Giorgini, Flaviano; Hollox, Edward J

2013-01-01

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of a CAG triplet repeat tract in the huntingtin gene. While the length of this CAG expansion is the major determinant of the age of onset (AO), other genetic factors have also been shown to play a modulatory role. Recent evidence suggests that neuroinflammations is a pivotal factor in the pathogenesis of HD, and that targeting this process may have important therapeutic ramifications. The human β-defensin 2 (hBD2)- encoded by DEFB4- is an antimicrobial peptide that exhibits inducible expression in astrocytes during inflammation and is an important regulator of innate and adaptive immune response. Therefore, DEFB4 may contribute to the neuroinflammatory processes observed in HD. In this study we tested the hypothesis that copy number variation (CNV) of the β-defensin region, including DEFB4, modifies the AO in HD. We genotyped β-defensin CNV in 490 HD individuals using the paralogue ratio test and found no association between β-defensin CNV and onset of HD. We conclude that it is unlikely that DEFB4 plays a role in HD pathogenesis.

Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas.

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Rosa, Rafael D; Santini, Adrien; Fievet, Julie; Bulet, Philippe; Destoumieux-Garzón, Delphine; Bachère, Evelyne

2011-01-01

Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms

Utilization of plant-derived recombinant human β-defensins (hBD-1 and hBD-2) for averting salmonellosis.

Science.gov (United States)

Patro, Sunita; Maiti, Soumitra; Panda, Santosh Kumar; Dey, Nrisingha

2015-04-01

We describe the use of plant-made β-defensins as effective antimicrobial substances for controlling salmonellosis, a deadly infection caused by Salmonella typhimurium (referred to further as S. typhi). Human β-defensin-1 (hBD-1) and -2 (hBD-2) were expressed under the control of strong constitutive promoters in tobacco plants, and bio-active β-defensins were successfully extracted. In the in vitro studies, enriched recombinant plant-derived human β-defensin-1 (phBD-1) and -2 (phBD-2) obtained from both T1 and T2 transgenic plants showed significant antimicrobial activity against Escherichia coli and S. typhi when used individually and in various combinations. The 2:1 peptide combination of phBD-1:phBD-2 with peptides isolated from T1-and T2-generation plants reduced the growth of S. typhi by 96 and 85 %, respectively. In vivo studies employing the mouse model (Balb/c) of Salmonella infection clearly demonstrated that the administration of plant-derived defensins individually and in different combinations enhanced the mean survival time of Salmonella-infected animals. When treatment consisted of the 2:1 phBD-1:phBD-2 combination, approximately 50 % of the infected mice were still alive at 206 h post-inoculation; the lowest number of viable S. typhi was observed in the liver and spleen of infected animals. We conclude that plant-made recombinant β-defensins (phBD-1 and phBD-2) are promising antimicrobial substances and have the potential to become additional tools against salmonellosis, particularly when used in combination.

Mode of action of plant defensins suggests therapeutic potential

NARCIS (Netherlands)

Thomma, B.P.H.J.; Cammue, B.P.A.; Thevissen, K.

2003-01-01

Higher vertebrates can rely both on an innate as well as an adaptive immune system for defense against invading pathogens. In contrast, plants can only employ an innate immune system that largely depends on the production of antimicrobial compounds such as plant defensins and other

Two novel antimicrobial defensins from rice identified by gene coexpression network analyses.

Science.gov (United States)

Tantong, Supaluk; Pringsulaka, Onanong; Weerawanich, Kamonwan; Meeprasert, Arthitaya; Rungrotmongkol, Thanyada; Sarnthima, Rakrudee; Roytrakul, Sittiruk; Sirikantaramas, Supaart

2016-10-01

Defensins form an antimicrobial peptides (AMP) family, and have been widely studied in various plants because of their considerable inhibitory functions. However, their roles in rice (Oryza sativa L.) have not been characterized, even though rice is one of the most important staple crops that is susceptible to damaging infections. Additionally, a previous study identified 598 rice genes encoding cysteine-rich peptides, suggesting there are several uncharacterized AMPs in rice. We performed in silico gene expression and coexpression network analyses of all genes encoding defensin and defensin-like peptides, and determined that OsDEF7 and OsDEF8 are coexpressed with pathogen-responsive genes. Recombinant OsDEF7 and OsDEF8 could form homodimers. They inhibited the growth of the bacteria Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Erwinia carotovora subsp. atroseptica with minimum inhibitory concentration (MIC) ranging from 0.6 to 63μg/mL. However, these OsDEFs are weakly active against the phytopathogenic fungi Helminthosporium oryzae and Fusarium oxysporum f.sp. cubense. This study describes a useful method for identifying potential plant AMPs with biological activities. Copyright © 2016 Elsevier Inc. All rights reserved.

Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

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Park Raekil

2006-01-01

Full Text Available Abstract Background We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi and that interleukin 1 alpha (IL-1 alpha up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. Methods The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM. Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. Results Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. Conclusion We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway.

Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

Science.gov (United States)

Rosa, Rafael D.; Santini, Adrien; Fievet, Julie; Bulet, Philippe; Destoumieux-Garzón, Delphine; Bachère, Evelyne

2011-01-01

Background Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Findings Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. Conclusions We provide here the first report showing that big defensins form a family of antimicrobial

Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas.

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Rafael D Rosa

Full Text Available BACKGROUND: Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. FINDINGS: Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3 that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. CONCLUSIONS: We provide here the first report showing that big defensins form a family

Ixodes ricinus defensins attack distantly-related pathogens

Czech Academy of Sciences Publication Activity Database

Tonk, M.; Cabezas-Cruz, A.; Valdés, James J.; Rego, Ryan O. M.; Grubhoffer, Libor; Estrada--Pena, A.; Vilcinskas, A.; Kotsyfakis, Michalis; Rahnamaeian, M.

2015-01-01

Roč. 53, č. 2 (2015), s. 358-365 ISSN 0145-305X R&D Projects: GA MŠk(CZ) EE2.3.30.0032; GA ČR GAP502/12/2409 EU Projects: European Commission(XE) 278976 Institutional support: RVO:60077344 Keywords : Antimicrobial peptide * Defensin * Ixodes ricinus * Listeria monocytogenes * Staphylococcus aureus * Staphylococcus epidermidis * Escherichia coli * Pseudomonas aeruginosa * Fusarium spp Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.620, year: 2015

An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.

Science.gov (United States)

Xu, X-X; Zhang, Y-Q; Freed, S; Yu, J; Gao, Y-F; Wang, S; Ouyang, L-N; Ju, W-Y; Jin, F-L

2016-12-01

Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

The β-Defensin Gallinacin-6 Is Expressed in the Chicken Digestive Tract and Has Antimicrobial Activity against Food-Borne Pathogens▿

Science.gov (United States)

van Dijk, Albert; Veldhuizen, Edwin J. A.; Kalkhove, Stefanie I. C.; Tjeerdsma-van Bokhoven, Johanna L. M.; Romijn, Roland A.; Haagsman, Henk P.

2007-01-01

Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of β-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken β-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16 μg/ml synthetic Gal-6 resulted in a 3 log unit reduction in Clostridium perfringens survival within 60 min, indicating fast killing kinetics. Transmission electron microscopy examination of synthetic-Gal-6-treated Clostridium perfringens cells showed dose-dependent changes in morphology after 30 min, including intracellular granulation, cytoplasm retraction, irregular septum formation in dividing cells, and cell lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest that chicken β-defensin gallinacin-6 plays an important role in chicken innate host defense. PMID:17194828

Determination of beta-defensin genomic copy number in different populations

DEFF Research Database (Denmark)

Fode, Peder; Jespersgaard, Cathrine; Hardwick, Robert J

2011-01-01

There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and ß-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with...

Tribolium castaneum defensins are primarily active against Gram-positive bacteria

Czech Academy of Sciences Publication Activity Database

Tonk, M.; Knorr, E.; Cabezas-Cruz, A.; Valdés, James J.; Kollewe, C.; Vilcinskas, A.

2015-01-01

Roč. 132, NOV 2015 (2015), s. 208-215 ISSN 0022-2011 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Antimicrobial peptides * Defensin * Innate immunity * Insects * Tribolium castaneum * Gram-positive bacteria Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.198, year: 2015

The host defense peptide beta-defensin 1 confers protection against Bordetella pertussis in newborn piglets.

Science.gov (United States)

Elahi, Shokrollah; Buchanan, Rachelle M; Attah-Poku, Sam; Townsend, Hugh G G; Babiuk, Lorne A; Gerdts, Volker

2006-04-01

Innate immunity plays an important role in protection against respiratory infections in humans and animals. Host defense peptides such as beta-defensins represent major components of innate immunity. We recently developed a novel porcine model of pertussis, an important respiratory disease of young children and infants worldwide. Here, we investigated the role of porcine beta-defensin 1 (pBD-1), a porcine defensin homologue of human beta-defensin 2, in conferring protection against respiratory infection with Bordetella pertussis. In this model, newborn piglets were fully susceptible to infection and developed severe bronchopneumonia. In contrast, piglets older than 4 weeks of age were protected against infection with B. pertussis. Protection was associated with the expression of pBD-1 in the upper respiratory tract. In fact, pBD-1 expression was developmentally regulated, and the absence of pBD-1 was thought to contribute to the increased susceptibility of newborn piglets to infection with B. pertussis. Bronchoalveolar lavage specimens collected from older animals as well as chemically synthesized pBD-1 displayed strong antimicrobial activity against B. pertussis in vitro. Furthermore, in vivo treatment of newborn piglets with only 500 mug pBD-1 at the time of challenge conferred protection against infection with B. pertussis. Interestingly, pBD-1 displayed no bactericidal activity in vitro against Bordetella bronchiseptica, a closely related natural pathogen of pigs. Our results demonstrate that host defense peptides play an important role in protection against pertussis and are essential in modulating innate immune responses against respiratory infections.

Generation of transgenic cattle expressing human β-defensin 3 as an approach to reducing susceptibility to Mycobacterium bovis infection.

Science.gov (United States)

Su, Feng; Wang, Yongsheng; Liu, Guanghui; Ru, Kun; Liu, Xin; Yu, Yuan; Liu, Jun; Wu, Yongyan; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2016-03-01

Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection. © 2016 Federation of European Biochemical Societies.

Proteomic Profile of Unstable Atheroma Plaque: Increased Neutrophil Defensin 1, Clusterin, and Apolipoprotein E Levels in Carotid Secretome.

Science.gov (United States)

Aragonès, Gemma; Auguet, Teresa; Guiu-Jurado, Esther; Berlanga, Alba; Curriu, Marta; Martinez, Salomé; Alibalic, Ajla; Aguilar, Carmen; Hernández, Esteban; Camara, María-Luisa; Canela, Núria; Herrero, Pol; Ruyra, Xavier; Martín-Paredero, Vicente; Richart, Cristóbal

2016-03-04

Because of the clinical significance of carotid atherosclerosis, the search for novel biomarkers has become a priority. The aim of the present study was to compare the protein secretion profile of the carotid atherosclerotic plaque (CAP, n = 12) and nonatherosclerotic mammary artery (MA, n = 10) secretomes. We used a nontargeted proteomic approach that incorporated tandem immunoaffinity depletion, iTRAQ labeling, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 162 proteins were quantified, of which 25 showed statistically significant differences in secretome levels between carotid atherosclerotic plaque and nondiseased mammary artery. We found increased levels of neutrophil defensin 1, apolipoprotein E, clusterin, and zinc-alpha-2-glycoprotein in CAP secretomes. Results were validated by ELISA assays. Also, differentially secreted proteins are involved in pathways such as focal adhesion and leukocyte transendothelial migration. In conclusion, this study provides a subset of identified proteins that are differently expressed in secretomes of clinical significance.

Expression of multidrug resistance proteins in retinoblastoma

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Swati Shukla

2017-11-01

Full Text Available AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1 expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

Expression of multidrug resistance proteins in retinoblastoma.

Science.gov (United States)

Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

2017-01-01

To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

Expression of multidrug resistance proteins in retinoblastoma

Science.gov (United States)

Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

2017-01-01

AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

Correlation of levels of alpha-defensins determined by HPLC-ESI-MS in bronchoalveolar lavage fluid with the diagnosis of pneumonia in premature neonates.

Science.gov (United States)

Tirone, Chiara; Boccacci, Simona; Inzitari, Rosanna; Tana, Milena; Aurilia, Claudia; Fanali, Chiara; Cabras, Tiziana; Messana, Irene; Castagnola, Massimo; Romagnoli, Costantino; Vento, Giovanni

2010-08-01

The presence of alpha-defensins in bronchoalveolar lavage fluid (BALF) was investigated in a cohort of preterm newborns with gestational age (GA) groups: pneumonia group of nine neonates suffering from pulmonary infection (GA: 26.1 +/- 2.1 wk; birth weight: 787.4 +/- 309.9 g), with or without associated bloodstream infection, and nonpneumonia group of 15 neonates (GA: 27.7 +/- 2.0 wk; birth weight: 1019.0 +/- 319.8 g). BALF culture was positive for CONS (n = 5), Staphylococcus aureus (n = 1), and Candida spp (n = 3). BALF samples were analyzed by HPLC-electrospray Ionization-mass spectrometer. The alpha-defensins 1-4 concentration, absolute and differential white cells count were measured. Relative amounts of alpha-defensins 1-4 and the absolute number of neutrophils were found significantly higher in the pneumonia group with respect to the nonpneumonia group (p < 0.05). Moreover, positive significant correlations between the number of neutrophils and the alpha-defensins 1-3 levels were observed. In conclusion, our data show that preterm newborns, also at the lower GA, are able to produce alpha-defensins, underlining that their innate defense system is already active before the at-term delivery date.

Bipolar resistive switching in different plant and animal proteins

KAUST Repository

Bag, A.; Hota, Mrinal Kanti; Mallik, Sandipan B.; Maì ti, Chinmay Kumar

2014-01-01

We report bipolar resistive switching phenomena observed in different types of plant and animal proteins. Using protein as the switching medium, resistive switching devices have been fabricated with conducting indium tin oxide (ITO) and Al as bottom and top electrodes, respectively. A clockwise bipolar resistive switching phenomenon is observed in all proteins. It is shown that the resistive switching phenomena originate from the local redox process in the protein and the ion exchange from the top electrode/protein interface.

Bipolar resistive switching in different plant and animal proteins

KAUST Repository

Bag, A.

2014-06-01

We report bipolar resistive switching phenomena observed in different types of plant and animal proteins. Using protein as the switching medium, resistive switching devices have been fabricated with conducting indium tin oxide (ITO) and Al as bottom and top electrodes, respectively. A clockwise bipolar resistive switching phenomenon is observed in all proteins. It is shown that the resistive switching phenomena originate from the local redox process in the protein and the ion exchange from the top electrode/protein interface.

Gene organization of a novel defensin of Ixodes ricinus: first annotation of an intron/exon structure in a hard tick defensin gene and first evidence of the occurrence of two isoforms of one member of the arthropod defensin family

Czech Academy of Sciences Publication Activity Database

Rudenko, Natalia; Golovchenko, Maryna; Grubhoffer, Libor

2007-01-01

Roč. 16, č. 4 (2007), s. 501-507 ISSN 0962-1075 R&D Projects: GA MŠk(CZ) LC06009; GA ČR(CZ) GA524/06/1479 Institutional research plan: CEZ:AV0Z60220518 Keywords : defensin * Ixodes ricinus * intron/exon structure * immune response * antimicrobial activity Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 2.787, year: 2007

Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

DEFF Research Database (Denmark)

Schneider, Tanja; Kruse, Thomas; Wimmer, Reinhard

2010-01-01

Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show th...

Novel phenotype of mouse spermatozoa following deletion of nine β-defensin genes.

Science.gov (United States)

Dorin, Julia R

2015-01-01

β-defensin peptides are a large family of antimicrobial peptides. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. Despite their inducible presence at mucosal surfaces, their main site of expression is the epididymis. Recent evidence suggests that a major function of these peptides is in sperm maturation. In addition to previous work suggesting this, work at the MRC Human Genetics Unit, Edinburgh, has shown that homozygous deletion of a cluster of nine β-defensin genes in the mouse results in profound male sterility. The spermatozoa derived from the mutants had reduced motility and increased fragility. Epididymal spermatozoa isolated from the cauda region of the homozygous mutants demonstrated precocious capacitation and increased spontaneous acrosome reactions compared with those from wild-types. Despite this, these mutant spermatozoa had reduced ability to bind to the zona pellucida of oocytes. Ultrastructural examination revealed a disintegration of the microtubule structure of mutant-derived spermatozoa isolated from the epididymal cauda region, but not from the caput. Consistent with premature acrosome reaction and hyperactivation, spermatozoa from mutant animals had significantly increased intracellular calcium content. This work demonstrates that in vivo β-defensins are essential for successful sperm maturation, and that their disruption alters intracellular calcium levels, which most likely leads to premature activation and spontaneous acrosome reactions that result in hyperactivation and loss of microtubule structure of the axoneme. Determining which of the nine genes are responsible for the phenotype and the relevance to human sperm function is important for future work on male infertility.

Antiplasmodial Activity Is an Ancient and Conserved Feature of Tick Defensins

Czech Academy of Sciences Publication Activity Database

Cabezas Cruz, Alejandro; Tonk, M.; Bouchut, A.; Pierrot, C.; Pierce, R.J.; Kotsyfakis, Michalis; Rahnamaeian, M.; Vilcinskas, A.; Khalife, J.; Valdés, James J.

2016-01-01

Roč. 7, 24 October (2016), č. článku 1682. ISSN 1664-302X R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : ticks * defensins * antimicrobial spectrum * ancestral sequence reconstruction * Plasmodium falciparum Subject RIV: EI - Biotechnology ; Bionics Impact factor: 4.076, year: 2016

Influence of Th2 Cytokines on the Cornified Envelope, Tight Junction Proteins, and ß-Defensins in Filaggrin-Deficient Skin Equivalents.

Science.gov (United States)

Hönzke, Stefan; Wallmeyer, Leonie; Ostrowski, Anja; Radbruch, Moritz; Mundhenk, Lars; Schäfer-Korting, Monika; Hedtrich, Sarah

2016-03-01

Atopic dermatitis is a chronic skin condition with complex etiology. It is characterized by skin barrier defects and T helper type 2 (Th2)-polarized inflammation. Although mutations in the filaggrin gene are known to be prominent genetic risk factors for the development of atopic dermatitis, the interdependency between these and an altered cytokine milieu is not fully understood. In this study, we evaluated the direct effects of filaggrin deficiency on the cornified envelope, tight junction proteins, and innate immune response, and report the effects of Th2 cytokines in normal and filaggrin-deficient skin equivalents. Supplementation with IL-4 and IL-13 led to distinct histologic changes and significantly increased skin surface pH, both of which were enhanced in filaggrin knockdown skin equivalents. We detected a compensatory up-regulation of involucrin and occludin in filaggrin-deficient skin that was dramatically disturbed when simultaneous inflammation occurred. Furthermore, we found that a lack of filaggrin triggered an up-regulation of human ?-defensin 2 via an unknown mechanism, which was abolished by Th2 cytokine supplementation. Taken together, these results indicate that defects in the epidermal barrier, skin permeability, and cutaneous innate immune response are not primarily linked to filaggrin deficiency but are rather secondarily induced by Th2 inflammation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Protein function prediction involved on radio-resistant bacteria

International Nuclear Information System (INIS)

Mezhoud, Karim; Mankai, Houda; Sghaier, Haitham; Barkallah, Insaf

2009-01-01

Previously, we identified 58 proteins under positive selection in ionizing-radiation-resistant bacteria (IRRB) but absent in all ionizing-radiation-sensitive bacteria (IRSB). These are good reasons to believe these 58 proteins with their interactions with other proteins (interactomes) are a part of the answer to the question as to how IRRB resist to radiation, because our knowledge of interactomes of positively selected orphan proteins in IRRB might allow us to define cellular pathways important to ionizing-radiation resistance. Using the Database of Interacting Proteins and the PSIbase, we have predicted interactions of orthologs of the 58 proteins under positive selection in IRRB but absent in all IRSB. We used integrate experimental data sets with molecular interaction networks and protein structure prediction from databases. Among these, 18 proteins with their interactomes were identified in Deinococcus radiodurans R1. DNA checkpoint and repair, kinases pathways, energetic and nucleotide metabolisms were the important biological process that found. We predicted the interactomes of 58 proteins under positive selection in IRRB. It is hoped our data will provide new clues as to the cellular pathways that are important for ionizing-radiation resistance. We have identified news proteins involved on DNA management which were not previously mentioned. It is an important input in addition to protein that studied. It does still work to deepen our study on these new proteins

Gastrointestinal Autoimmunity Associated With Loss of Central Tolerance to Enteric alpha-Defensins

Czech Academy of Sciences Publication Activity Database

Dobeš, Jan; Neuwirth, Aleš; Dobešová, Martina; Vobořil, Matouš; Balounová, Jana; Ballek, Ondřej; Lebl, J.; Meloni, A.; Krohn, K.; Kluger, N.; Ranki, A.; Filipp, Dominik

2015-01-01

Roč. 149, č. 1 (2015), s. 139-150 ISSN 0016-5085 R&D Projects: GA ČR(CZ) GBP302/12/G101 Institutional support: RVO:68378050 Keywords : Enteric defensins * Intestinal autoimmunity * Mouse Model of APECED Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 18.187, year: 2015

Proteinase 3 carries small unusual carbohydrates and associates with αlpha-defensins

DEFF Research Database (Denmark)

Zoega, Morten; Ravnsborg, Tina; Højrup, Peter

2012-01-01

with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with α-defensins and oligomers hereof. Irreversible inhibition of PR3 by α1-antitrypsin did not affect its association...

Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: Identification of an RGFRRR motif governing fungal cell entry

Energy Technology Data Exchange (ETDEWEB)

Sagaram, Uma S.; El-Mounadi, Kaoutar; Buchko, Garry W.; Berg, Howard R.; Kaur, Jagdeep; Pandurangi, Raghoottama; Smith, Thomas J.; Shah, Dilip

2013-12-04

A highly conserved plant defensin MtDef4 potently inhibits the growth of a filamentous fungus Fusarium graminearum. MtDef4 is internalized by cells of F. graminearum. To determine its mechanism of fungal cell entry and antifungal action, NMR solution structure of MtDef4 has been determined. The analysis of its structure has revealed a positively charged patch on the surface of the protein consisting of arginine residues in its γ-core signature, a major determinant of the antifungal activity of MtDef4. Here, we report functional analysis of the RGFRRR motif of the γ-core signature of MtDef4. The replacement of RGFRRR to AAAARR or to RGFRAA not only abolishes fungal cell entry but also results in loss of the antifungal activity of MtDef4. MtDef4 binds strongly to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Mutations of RGFRRR which abolish fungal cell entry of MtDef4 also impair its binding to PA. Our results suggest that RGFRRR motif is a translocation signal for entry of MtDef4 into fungal cells and that this positively charged motif likely mediates interaction of this defensin with PA as part of its antifungal action.

Novel phenotype of mouse spermatozoa following deletion of nine β-defensin genes

Directory of Open Access Journals (Sweden)

Julia R Dorin

2015-01-01

Full Text Available β-defensin peptides are a large family of antimicrobial peptides. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. Despite their inducible presence at mucosal surfaces, their main site of expression is the epididymis. Recent evidence suggests that a major function of these peptides is in sperm maturation. In addition to previous work suggesting this, work at the MRC Human Genetics Unit, Edinburgh, has shown that homozygous deletion of a cluster of nine β-defensin genes in the mouse results in profound male sterility. The spermatozoa derived from the mutants had reduced motility and increased fragility. Epididymal spermatozoa isolated from the cauda region of the homozygous mutants demonstrated precocious capacitation and increased spontaneous acrosome reactions compared with those from wild-types. Despite this, these mutant spermatozoa had reduced ability to bind to the zona pellucida of oocytes. Ultrastructural examination revealed a disintegration of the microtubule structure of mutant-derived spermatozoa isolated from the epididymal cauda region, but not from the caput. Consistent with premature acrosome reaction and hyperactivation, spermatozoa from mutant animals had significantly increased intracellular calcium content. This work demonstrates that in vivo β-defensins are essential for successful sperm maturation, and that their disruption alters intracellular calcium levels, which most likely leads to premature activation and spontaneous acrosome reactions that result in hyperactivation and loss of microtubule structure of the axoneme. Determining which of the nine genes are responsible for the phenotype and the relevance to human sperm function is important for future work on male infertility.

Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: identification of an RGFRRR motif governing fungal cell entry.

Directory of Open Access Journals (Sweden)

Uma Shankar Sagaram

Full Text Available MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β2 and β3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA, a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.

Expression and new exon mutations of the human Beta defensins and their association on colon cancer development.

Directory of Open Access Journals (Sweden)

Abdelhabib Semlali

Full Text Available The development of cancer involves genetic predisposition and a variety of environmental exposures. Genome-wide linkage analyses provide evidence for the significant linkage of many diseases to susceptibility loci on chromosome 8p23, the location of the human defensin gene cluster. Human β-defensins (hBDs are important molecules of innate immunity. This study was designed to analyze the expression and genetic variations in hBDs (hBD-1, hBD-2, hBD-3 and hBD-4 and their putative association with colon cancer. hBD gene expression and relative protein expression were evaluated by Real-Time polymerase chain reaction (qPCR and immunohistochemistry, respectively, from 40 normal patients and 40 age-matched patients with colon cancer in Saudi Arabia. In addition, hBD polymorphisms were genotyped by exon sequencing and by promoter methylation. hBD-1, hBD-2, hBD-3 and hBD-4 basal messenger RNA expression was significantly lower in tumor tissues compared with normal tissues. Several insertion mutations were detected in different exons of the analyzed hBDs. However, no methylation in any hBDs promoters was detected because of the limited number of CpG islands in these regions. We demonstrated for the first time a link between hBD expression and colon cancer. This suggests that there is a significant link between innate immunity deregulation through disruption of cationic peptides (hBDs and the potential development of colon cancer.

Multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP and lung resistance protein (LRP gene expression in childhood acute lymphoblastic leukemia

Directory of Open Access Journals (Sweden)

Elvis Terci Valera

Full Text Available CONTEXT: Despite the advances in the cure rate for acute lymphoblastic leukemia, approximately 25% of affected children suffer relapses. Expression of genes for the multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP, and lung resistance protein (LRP may confer the phenotype of resistance to the treatment of neoplasias. OBJECTIVE: To analyze the expression of the MDR-1, MRP and LRP genes in children with a diagnosis of acute lymphoblastic leukemia via the semiquantitative reverse transcription polymerase chain reaction (RT-PCR, and to determine the correlation between expression and event-free survival and clinical and laboratory variables. DESIGN: A retrospective clinical study. SETTING: Laboratory of Pediatric Oncology, Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil. METHODS: Bone marrow aspirates from 30 children with a diagnosis of acute lymphoblastic leukemia were assessed for the expression of messenger RNA for the MDR-1, MRP and LRP genes by semi-quantitative RT-PCR. RESULTS: In the three groups studied, only the increased expression of LRP was related to worsened event-free survival (p = 0.005. The presence of the common acute lymphoblastic leukemia antigen (CALLA was correlated with increased LRP expression (p = 0.009 and increased risk of relapse or death (p = 0.05. The relative risk of relapse or death was six times higher among children with high LRP expression upon diagnosis (p = 0.05, as confirmed by multivariate analysis of the three genes studied (p = 0.035. DISCUSSION: Cell resistance to drugs is a determinant of the response to chemotherapy and its detection via RT-PCR may be of clinical importance. CONCLUSIONS: Evaluation of the expression of genes for resistance to antineoplastic drugs in childhood acute lymphoblastic leukemia upon diagnosis, and particularly the expression of the LRP gene, may be of clinical relevance, and should be the

α-Defensins Induce a Post-translational Modification of Low Density Lipoprotein (LDL) That Promotes Atherosclerosis at Normal Levels of Plasma Cholesterol.

Science.gov (United States)

Abu-Fanne, Rami; Maraga, Emad; Abd-Elrahman, Ihab; Hankin, Aviel; Blum, Galia; Abdeen, Suhair; Hijazi, Nuha; Cines, Douglas B; Higazi, Abd Al-Roof

2016-02-05

Approximately one-half of the patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating that additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition, and atherogenesis using transgenic mice expressing human α-defensins in their polymorphonuclear leukocytes (Def(+/+)). Accelerated Def(+/+) mice developed α-defensin·LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL, and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def(+/+) to WT mice increased LDL clearance, increased vascular permeability, and increased vascular deposition of LDL, whereas transplantation of WT bone marrow to Def(+/+) mice prevented these outcomes. The same outcome was obtained by treating Def(+/+) mice with colchicine to inhibit the release of α-defensins. These studies identify a potential new link between inflammation and the development of atherosclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PaDef defensin from avocado (Persea americana var. drymifolia) is cytotoxic to K562 chronic myeloid leukemia cells through extrinsic apoptosis.

Science.gov (United States)

Flores-Alvarez, Luis José; Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Salgado-Garciglia, Rafael; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2018-06-01

Plant defensins, a group of antimicrobial peptides, show selective cytotoxicity toward cancer cells. However, their mechanisms of action remain poorly understood. Here, we evaluated the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on K562 chronic myeloid leukemia cells and analyzed the pathway involved in the induction of cell death. The defensin PaDef was not cytotoxic against human PBMCs; however, it was cytotoxic for K562 cell line (IC 50  = 97.3 μg/ml) activating apoptosis at 12 h. PaDef did not affect the mitochondrial membrane potential (ΔΨm), neither the transmembranal potential or the release of intracellular calcium. Also, PaDef induced gene expression of caspase 8 (∼2 fold), TNF-α (∼4 fold) and TNFR1 (∼10 fold). In addition, the activation of caspase 8 was detected at 24 h, whereas caspase 9 activity was not modified, suggesting that the extrinsic apoptosis pathway could be activated. In conclusion, PaDef induces apoptosis on K562 cells, which is related to the activation of caspase 8 and involves the participation of TNF-α, which is a novel property for a plant defensin. Copyright © 2018 Elsevier Ltd. All rights reserved.

Partial deletion of chromosome 8 β-defensin cluster confers sperm dysfunction and infertility in male mice.

Directory of Open Access Journals (Sweden)

Yu S Zhou

2013-10-01

Full Text Available β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9 in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.

The Role of α-Defensins 1–3 in Antimicrobial Protection Forming in Children with Recurrent Bronchitis Caused by Bacteria of the Genus Haemophilus

Directory of Open Access Journals (Sweden)

G.O. Lezhenko

2013-03-01

Full Text Available The level of α-defensins 1–3 (HNP 1–3 has been analyzed in the blood plasma of children with recurrent bronchitis caused by bacteria of the genus Haemophilus. It is shown that the level of HNP 1–3 in the blood plasma depends on the form of Haemophilus. Trigger of HNP 1–3 outflow for neutrophils was the presence of bacterial capsule while presence of L-forms of Haemophilus influenzae wasn’t associated with increase in synthesis of antimicrobial peptides that could be one of the factors of forming of Haemophilus antibiotic resistance.

Human α-defensin (DEFA) gene expression helps to characterise benign and malignant salivary gland tumours

International Nuclear Information System (INIS)

Winter, Jochen; Wenghoefer, Matthias; Pantelis, Annette; Kraus, Dominik; Reckenbeil, Jan; Reich, Rudolf; Jepsen, Soeren; Fischer, Hans-Peter; Allam, Jean-Pierre; Novak, Natalija

2012-01-01

Because of the infrequence of salivary gland tumours and their complex histopathological diagnosis it is still difficult to exactly predict their clinical course by means of recurrence, malignant progression and metastasis. In order to define new proliferation associated genes, purpose of this study was to investigate the expression of human α-defensins (DEFA) 1/3 and 4 in different tumour entities of the salivary glands with respect to malignancy. Tissue of salivary glands (n=10), pleomorphic adenomas (n=10), cystadenolymphomas (n=10), adenocarcinomas (n=10), adenoidcystic carcinomas (n=10), and mucoepidermoid carcinomas (n=10) was obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of DEFA 1/3 and 4 were analyzed by quantitative realtime PCR and compared with healthy salivary gland tissue. Additionally, the proteins encoded by DEFA 1/3 and DEFA 4 were visualized in paraffin-embedded tissue sections by immunohistochemical staining. Human α-defensins are traceable in healthy as well as in pathological altered salivary gland tissue. In comparison with healthy tissue, the gene expression of DEFA 1/3 and 4 was significantly (p<0.05) increased in all tumours – except for a significant decrease of DEFA 4 gene expression in pleomorphic adenomas and a similar transcript level for DEFA 1/3 compared to healthy salivary glands. A decreased gene expression of DEFA 1/3 and 4 might protect pleomorphic adenomas from malignant transformation into adenocarcinomas. A similar expression pattern of DEFA-1/3 and -4 in cystadenolymphomas and inflamed salivary glands underlines a potential importance of immunological reactions during the formation of Warthin’s tumour

Receptor-like proteins involved in plant disease resistance

NARCIS (Netherlands)

Kruijt, M.; Kock, de M.J.D.; Wit, de P.J.G.M.

2005-01-01

Race-specific resistance in plants against microbial pathogens is governed by several distinct classes of resistance (R) genes. This review focuses on the class that consists of the plasma membrane-bound leucine-rich repeat proteins known as receptor-like proteins (RLPs). The first isolated

Lucifensins, the Insect Defensins of Biomedical Importance: The Story behind Maggot Therapy

Czech Academy of Sciences Publication Activity Database

Čeřovský, Václav; Bém, R.

2014-01-01

Roč. 7, č. 3 (2014), s. 251-264 ISSN 1424-8247 R&D Projects: GA ČR GA203/08/0536 Institutional support: RVO:61388963 Keywords : antimicrobial peptide * insect defensin * lucifensin * maggot therapy * Lucilia sericata * Lucilia cuprina * peptide isolation * peptide identification Subject RIV: CC - Organic Chemistry http://www.mdpi.com/1424-8247/7/3/251

ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

Science.gov (United States)

Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

2016-03-22

Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

Microwave processing of honey negatively affects honey antibacterial activity by inactivation of bee-derived glucose oxidase and defensin-1.

Science.gov (United States)

Bucekova, Marcela; Juricova, Valeria; Monton, Enrique; Martinotti, Simona; Ranzato, Elia; Majtan, Juraj

2018-02-01

Microwave (MW) thermal heating has been proposed as an efficient method for honey liquefaction, while maintaining honey quality criteria. However, little is known about the effects of MW thermal heating on honey antibacterial activity. In this study, we aimed to determine the effects of MW heating on the antibacterial activity of raw rapeseed honeys against Pseudomonas aeruginosa and Staphylococcus aureus, with a particular focus on two major bee-derived antibacterial components, defensin-1 and hydrogen peroxide (H 2 O 2 ). Our results demonstrated that MW thermal heating completely abolished honey antibacterial activity whereas conventional thermal treatment at 45 and 55°C did not affect the antibacterial activity of honey samples. A significant decrease in both glucose oxidase activity and H 2 O 2 production as well as defensin-1 amount was observed in MW-treated samples. Given that defensin-1 and H 2 O 2 are regular antibacterial components of all honeys, MW heating may have similar negative effects on every type of crystallized/liquid honey. Copyright © 2017 Elsevier Ltd. All rights reserved.

Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines

NARCIS (Netherlands)

Scheffer, GL; Maliepaard, M; Pijnenborg, ACLM; van Gastelen, MA; Schroeijers, AB; Allen, JD; Ross, DD; van der Valk, P; Dalton, WS; Schellens, JHM; Scheper, RJ; de Jong, MC

2000-01-01

Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (,MDR1) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported

Association of beta-Defensin Copy Number and Psoriasis in Three Cohorts of European Origin

NARCIS (Netherlands)

Stuart, P.E.; Huffmeier, U.; Nair, R.P.; Palla, R.; Tejasvi, T.; Schalkwijk, J.; Elder, J.T.; Reis, A.; Armour, J.A.

2012-01-01

A single previous study has demonstrated significant association of psoriasis with copy number of beta-defensin genes, using DNA from psoriasis cases and controls from Nijmegen and Erlangen. In this study, we attempted to replicate that finding in larger new cohorts from Erlangen (N=2,017) and

Oviduct-Specific Expression of Human Neutrophil Defensin 4 in Lentivirally Generated Transgenic Chickens

Science.gov (United States)

Liu, Tongxin; Wu, Hanyu; Cao, Dainan; Li, Qingyuan; Zhang, Yaqiong; Li, Ning; Hu, Xiaoxiang

2015-01-01

The expression of oviduct-specific recombinant proteins in transgenic chickens is a promising technology for the production of therapeutic biologics in eggs. In this study, we constructed a lentiviral vector encoding an expression cassette for human neutrophil defensin 4 (HNP4), a compound that displays high activity against Escherichia coli, and produced transgenic chickens that expressed the recombinant HNP4 protein in egg whites. After the antimicrobial activity of the recombinant HNP4 protein was tested at the cellular level, a 2.8-kb ovalbumin promoter was used to drive HNP4 expression specifically in oviduct tissues. From 669 injected eggs, 218 chickens were successfully hatched. Ten G0 roosters, with semens identified as positive for the transgene, were mated with wild-type hens to generate G1 chickens. From 1,274 total offspring, fifteen G1 transgenic chickens were positive for the transgene, which was confirmed by PCR and Southern blotting. The results of the Southern blotting and genome walking indicated that a single copy of the HNP4 gene was integrated into chromosomes 1, 2, 3, 4, 6 and 24 of the chickens. As expected, HNP4 expression was restricted to the oviduct tissues, and the levels of both transcriptional and translational HNP4 expression varied greatly in transgenic chickens with different transgene insertion sites. The amount of HNP4 protein expressed in the eggs of G1 and G2 heterozygous transgenic chickens ranged from 1.65 μg/ml to 10.18 μg/ml. These results indicated that the production of transgenic chickens that expressed HNP4 protein in egg whites was successful. PMID:26020529

Allelic recombination between distinct genomic locations generates copy number diversity in human β-defensins

Science.gov (United States)

Bakar, Suhaili Abu; Hollox, Edward J.; Armour, John A. L.

2009-01-01

β-Defensins are small secreted antimicrobial and signaling peptides involved in the innate immune response of vertebrates. In humans, a cluster of at least 7 of these genes shows extensive copy number variation, with a diploid copy number commonly ranging between 2 and 7. Using a genetic mapping approach, we show that this cluster is at not 1 but 2 distinct genomic loci ≈5 Mb apart on chromosome band 8p23.1, contradicting the most recent genome assembly. We also demonstrate that the predominant mechanism of change in β-defensin copy number is simple allelic recombination occurring in the interval between the 2 distinct genomic loci for these genes. In 416 meiotic transmissions, we observe 3 events creating a haplotype copy number not found in the parent, equivalent to a germ-line rate of copy number change of ≈0.7% per gamete. This places it among the fastest-changing copy number variants currently known. PMID:19131514

Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed

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Schininà Maria

2010-09-01

Full Text Available Abstract Background Several mutations have been described as responsible for rifampicin resistance in Neisseria meningitidis. However, the intriguing question on why these strains are so rare remains open. The aim of this study was to investigate the protein content and to identify differential expression in specific proteins in two rifampicin resistant and one susceptible meningococci using two-dimensional electrophoresis (2-DE combined with mass spectrometry. Results In our experimental conditions, able to resolve soluble proteins with an isoelectric point between 4 and 7, twenty-three proteins have been found differentially expressed in the two resistant strains compared to the susceptible. Some of them, involved in the main metabolic pathways, showed an increased expression, mainly in the catabolism of pyruvate and in the tricarboxylic acid cycle. A decreased expression of proteins belonging to gene regulation and to those involved in the folding of polypeptides has also been observed. 2-DE analysis showed the presence of four proteins displaying a shift in their isoelectric point in both resistant strains, confirmed by the presence of amino acid changes in the sequence analysis, absent in the susceptible. Conclusions The analysis of differentially expressed proteins suggests that an intricate series of events occurs in N. meningitidis rifampicin resistant strains and the results here reported may be considered a starting point in understanding their decreased invasion capacity. In fact, they support the hypothesis that the presence of more than one protein differentially expressed, having a role in the metabolism of the meningococcus, influences its ability to infect and to spread in the population. Different reports have described and discussed how a drug resistant pathogen shows a high biological cost for survival and that may also explain why, for some pathogens, the rate of resistant organisms is relatively low considering the

Loop Replacement Enhances the Ancestral Antibacterial Function of a Bifunctional Scorpion Toxin

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Shangfei Zhang

2018-06-01

Full Text Available On the basis of the evolutionary relationship between scorpion toxins targeting K+ channels (KTxs and antibacterial defensins (Zhu S., Peigneur S., Gao B., Umetsu Y., Ohki S., Tytgat J. Experimental conversion of a defensin into a neurotoxin: Implications for origin of toxic function. Mol. Biol. Evol. 2014, 31, 546–559, we performed protein engineering experiments to modify a bifunctional KTx (i.e., weak inhibitory activities on both K+ channels and bacteria via substituting its carboxyl loop with the structurally equivalent loop of contemporary defensins. As expected, the engineered peptide (named MeuTXKα3-KFGGI remarkably improved the antibacterial activity, particularly on some Gram-positive bacteria, including several antibiotic-resistant opportunistic pathogens. Compared with the unmodified toxin, its antibacterial spectrum also enlarged. Our work provides a new method to enhance the antibacterial activity of bifunctional scorpion venom peptides, which might be useful in engineering other proteins with an ancestral activity.

Resistance training reduces whole-body protein turnover and improves net protein retention in untrained young males.

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Hartman, Joseph W; Moore, Daniel R; Phillips, Stuart M

2006-10-01

It is thought that resistance exercise results in an increased need for dietary protein; however, data also exists to support the opposite conclusion. The purpose of this study was to determine the impact of resistance exercise training on protein metabolism in novices with the hypothesis that resistance training would reduce protein turnover and improve whole-body protein retention. Healthy males (n = 8, 22 +/- 1 y, BMI = 25.3 +/- 1.8 kg.m(-2)) participated in a progressive whole-body split routine resistance-training program 5d/week for 12 weeks. Before (PRE) and after (POST) the training, oral [15N]-glycine ingestion was used to assess nitrogen flux (Q), protein synthesis (PS), protein breakdown (PB), and net protein balance (NPB = PS-PB). Macronutrient intake was controlled over a 5d period PRE and POST, while estimates of protein turnover and urinary nitrogen balance (N(bal) = N(in) - urine N(out)) were conducted. Bench press and leg press increased 40% and 50%, respectively (p training-induced increases in both NPB (PRE = 0.22 +/- 0.13 g.kg(-1).d(-1); POST = 0.54 +/- 0.08 g.kg(-1).d(-1)) and urinary nitrogen balance (PRE = 2.8 +/- 1.7 g N.d(-1); POST = 6.5 +/- 0.9 g N.d(-1)) were observed. A program of resistance training that induced significant muscle hypertrophy resulted in reductions of both whole-body PS and PB, but an improved NPB, which favoured the accretion of skeletal muscle protein. Urinary nitrogen balance increased after training. The reduction in PS and PB and a higher NPB in combination with an increased nitrogen balance after training suggest that dietary requirements for protein in novice resistance-trained athletes are not higher, but lower, after resistance training.

Identification and partial characterisation of new members of the Ixodes ricinus defensin family

Czech Academy of Sciences Publication Activity Database

Tonk, Miray; Cabezas Cruz, Alejandro; Valdés, James J.; Rego, Ryan O. M.; Rudenko, Natalia; Golovchenko, Maryna; Bell-Sakyi, L.; de la Fuente, J.; Grubhoffer, Libor

2014-01-01

Roč. 540, č. 2 (2014), s. 146-152 ISSN 0378-1119 R&D Projects: GA ČR(CZ) GAP302/11/1901; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : antimicrobial peptide * defensin * Ixodes ricinus * tick * tick cell line Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.138, year: 2014

Expression of beta-defensins pBD-1 and pBD-2 along the small tract of the pig: Lack of upregulation in vivo upon Salmonella typhimurium infection

NARCIS (Netherlands)

Veldhuizen, E.J.; Dijk, van A.; Tersteeg, M.H.; Kalkhove, S.I.; Meulen, van der J.; Niewold, T.A.; Haagsman, H.P.

2007-01-01

Defensins are antimicrobial peptides that play an important role in the innate immune response in the intestine. Up to date, only one ß-defensin (pBD-1), has been described in pig, which was found to be expressed at low levels in the intestine. We set-up a quantitative PCR method to detect the gene

Expression of beta-defensins pBD-1 and pBD-2 along the small intestinal tract of the pig: lack of upregulation in vivo upon Salmonella typhimurium infection.

Science.gov (United States)

Veldhuizen, Edwin J A; van Dijk, Albert; Tersteeg, Monique H G; Kalkhove, Stefanie I C; van der Meulen, Jan; Niewold, Theo A; Haagsman, Henk P

2007-01-01

Defensins are antimicrobial peptides that play an important role in the innate immune response in the intestine. Up to date, only one beta-defensin (pBD-1), has been described in pig, which was found to be expressed at low levels in the intestine. We set-up a quantitative PCR method to detect the gene expression of pBD-1 and a newly discovered porcine beta-defensin, pBD-2. Expression of pBD-1 mRNA increased from the proximal to the distal part of the intestine whereas pBD-2 expression decreased. The main gene expression sites for pBD-2 were kidney and liver, whereas pBD-1 was mainly expressed in tongue. The porcine small intestinal segment perfusion (SISP) technique was used to investigate effects of Salmonella typhimurium DT104 on intestinal morphology and pBD-1 and pBD-2 mRNA levels in vivo. The early responses were studied 2, 4 and 8 h post-infection in four separate jejunal and ileal segments. Immunohistochemistry showed invasion of the mucosa by Salmonella and changes in intestinal morphology. However, no concomitant changes in expression of either pBD-1 or pBD-2 were observed. We conclude that at least two defensins are differentially expressed in the intestine of pigs, and that expression of both defensins is not altered by S. typhimurium under these conditions.

Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance.

Science.gov (United States)

Ali, Sajad; Ganai, Bashir Ahmad; Kamili, Azra N; Bhat, Ajaz Ali; Mir, Zahoor Ahmad; Bhat, Javaid Akhter; Tyagi, Anshika; Islam, Sheikh Tajamul; Mushtaq, Muntazir; Yadav, Prashant; Rawat, Sandhya; Grover, Anita

Pathogenesis-related (PR) proteins and antimicrobial peptides (AMPs) are a group of diverse molecules that are induced by phytopathogens as well as defense related signaling molecules. They are the key components of plant innate immune system especially systemic acquired resistance (SAR), and are widely used as diagnostic molecular markers of defense signaling pathways. Although, PR proteins and peptides have been isolated much before but their biological function remains largely enigmatic despite the availability of new scientific tools. The earlier studies have demonstrated that PR genes provide enhanced resistance against both biotic and abiotic stresses, which make them one of the most promising candidates for developing multiple stress tolerant crop varieties. In this regard, plant genetic engineering technology is widely accepted as one of the most fascinating approach to develop the disease resistant transgenic crops using different antimicrobial genes like PR genes. Overexpression of PR genes (chitinase, glucanase, thaumatin, defensin and thionin) individually or in combination have greatly uplifted the level of defense response in plants against a wide range of pathogens. However, the detailed knowledge of signaling pathways that regulates the expression of these versatile proteins is critical for improving crop plants to multiple stresses, which is the future theme of plant stress biology. Hence, this review provides an overall overview on the PR proteins like their classification, role in multiple stresses (biotic and abiotic) as well as in various plant defense signaling cascades. We also highlight the success and snags of transgenic plants expressing PR proteins and peptides. Copyright © 2018 Elsevier GmbH. All rights reserved.

Drug efflux proteins in multidrug resistant bacteria

NARCIS (Netherlands)

vanVeen, HW; Konings, WN

Bacteria contain an array of transport proteins in their cytoplasmic membrane. Many of these proteins play an important role in conferring resistance to toxic compounds. The multidrug efflux systems encountered in prokaryotic cells are very similar to those observed in eukaryotic cells. Therefore, a

[Study on vaginal production of human defensins and the correlated pathogenetic factors of vulvovaginal candidiasis].

Science.gov (United States)

Wang, Wen; DI, Wen; Liao, Qin-ping; Liu, Zhao-hui; Zhang, Ning; Zhang, Hui-ying; Zhang, Dai; Geng, Li; Fan, Shang-rong; Hu, Li-na

2008-07-01

To investigate the correlated pathogenetic factors and vaginal local immunity in vulvovaginal candidiasis (VVC). A case control study was conducted to compare VVC group (60 cases) with normal group (60 cases). All of the women filled up the specific questionnaires. Routine examination, pH test and bacterial culture were done on the vaginal discharge. Cytokines of the vaginal lavage were measured by enzyme linked immunosorbent assay. (1) Outcomes of the questionnaires: there was no significant difference between the two groups in educational background, knowledge of gynecologic infection, history of gynecologic infection, hygienic habit, sex life, or use of medicine (P > 0.05). The incidence of chronic cervicitis in normal group (43%, 26/60) was higher than in VVC group (22%, 13/60; P vaginal pH between the two groups (P > 0.05). (3) Detection rate of candida albicans by vaginal discharge routine examination was 72% (43/60). (4) The concentrations of interleukin (IL) 2, and IL-4 in vaginal lavage did not show significant difference between the two groups (P > 0.05), but the concentrations of human defensin 5, human beta-defensin (HBD) 1, and HBD2 in VVC group [(0.94 +/- 0.44) mg/L, (3.1 +/- 0.4) microg/L, (10 +/- 6) microg/L] were higher than normal group (P < 0.05). VVC is a common vulvovaginitis. There is no significant correlation between the incidence of VVC and educational background, knowledge of gynecologic infection, history of gynecologic infection, hygienic habit, sex life, or use of medicine in the child-bearing period. Human defensin may be closely correlated with the pathogenesis of VVC.

Prevalence of Resistence to Activated Protein C (Apc-Resistance in Blood Donors in Kosovo

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Ymer Mekaj

2009-11-01

Full Text Available AbstractOne of the most frequent hereditary causes of thrombophilia is, without a doubt, resistance to Activated Protein C (APC-resistance, which is a consequence of point mutation in gene coding for coagulation Factor V (Factor V Leiden in 90-95% of cases.The aim of this paper was to determine prevalence of APC-resistance in a group of healthy blood donors. The size of the group is quite representative of Kosovo Albanians.A total of 944 blood donors were examined (537 males and 407 females, for whom APC-resistance was determined by functional methods of coagulation using the kit ACTICLOT® Protein C Resistance. Method is based on the test of APTT determined twice: first in the presence and second in the absence of activated Protein C (APC. The ratio of these two values constitutes is called Activated Protein C - Sensitivity Ratio (APC-SR.From 944 examined donors, pathologic values of APC-SR (1,3-1,9 were found in 32 persons (3,4% of the total number. The distribution among sexes was 3,35% (18/537 in male and 3,43% (14/407 in female subjects. The mean values of APC-SR (1,64 in male and 1,71 in female subjects were not significantly different (P = 0,22.Based on these results, we conclude that the prevalence of APC resistance in Albanian population of Kosovo is within the lower limit of prevalence in general population in different countries of European countries, which, according to some authors ranges is from 3 to 7%.

Dietary protein to maximize resistance training: a review and examination of protein spread and change theories.

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Bosse, John D; Dixon, Brian M

2012-09-08

An appreciable volume of human clinical data supports increased dietary protein for greater gains from resistance training, but not all findings are in agreement. We recently proposed "protein spread theory" and "protein change theory" in an effort to explain discrepancies in the response to increased dietary protein in weight management interventions. The present review aimed to extend "protein spread theory" and "protein change theory" to studies examining the effects of protein on resistance training induced muscle and strength gains. Protein spread theory proposed that there must have been a sufficient spread or % difference in g/kg/day protein intake between groups during a protein intervention to see muscle and strength differences. Protein change theory postulated that for the higher protein group, there must be a sufficient change from baseline g/kg/day protein intake to during study g/kg/day protein intake to see muscle and strength benefits. Seventeen studies met inclusion criteria. In studies where a higher protein intervention was deemed successful there was, on average, a 66.1% g/kg/day between group intake spread versus a 10.2% g/kg/day spread in studies where a higher protein diet was no more effective than control. The average change in habitual protein intake in studies showing higher protein to be more effective than control was +59.5% compared to +6.5% when additional protein was no more effective than control. The magnitudes of difference between the mean spreads and changes of the present review are similar to our previous review on these theories in a weight management context. Providing sufficient deviation from habitual intake appears to be an important factor in determining the success of additional protein in enhancing muscle and strength gains from resistance training. An increase in dietary protein favorably effects muscle and strength during resistance training.

Functional characterization of two defensin isoforms of the hard tick Ixodes ricinus

Czech Academy of Sciences Publication Activity Database

Chrudimská, Tereza; Slaninová, Jiřina; Rudenko, Natalia; Růžek, Daniel; Grubhoffer, Libor

2011-01-01

Roč. 4, č. 1 (2011), e63 ISSN 1756-3305 R&D Projects: GA MŠk(CZ) LC06009; GA ČR GA206/09/1782; GA ČR GD206/09/H026; GA ČR(CZ) GAP302/11/1901 Institutional research plan: CEZ:AV0Z60220518; CEZ:AV0Z40550506 Keywords : defensin * antimicrobial compounds * Ixodes ricinus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.937, year: 2011

The mycorrhiza-dependent defensin MtDefMd1 of Medicago truncatula acts during the late restructuring stages of arbuscule-containing cells.

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Marian Uhe

Full Text Available Different symbiotic and pathogenic plant-microbe interactions involve the production of cysteine-rich antimicrobial defensins. In Medicago truncatula, the expression of four MtDefMd genes, encoding arbuscular mycorrhiza-dependent defensins containing an N-terminal signal peptide and exhibiting some differences to non-symbiotic defensins, raised over the time of fungal colonization. Whereas the MtDefMd1 and MtDefMd2 promoters were inactive in cells containing young arbuscules, cells with fully developed arbuscules displayed different levels of promoter activities, indicating an up-regulation towards later stages of arbuscule formation. MtDefMd1 and MtDefMd2 expression was absent or strongly down-regulated in mycorrhized ram1-1 and pt4-2 mutants, known for defects in arbuscule branching or premature arbuscule degeneration, respectively. A ~97% knock-down of MtDefMd1/MtDefMd2 expression did not significantly affect arbuscule size. Although overexpression of MtDefMd1 in arbuscule-containing cells led to an up-regulation of MtRam1, encoding a key transcriptional regulator of arbuscule formation, no morphological changes were evident. Co-localization of an MtDefMd1-mGFP6 fusion with additional, subcellular markers revealed that this defensin is associated with arbuscules in later stages of their life-cycle. MtDefMd1-mGFP6 was detected in cells with older arbuscules about to collapse, and ultimately in vacuolar compartments. Comparisons with mycorrhized roots expressing a tonoplast marker indicated that MtDefMd1 acts during late restructuring processes of arbuscule-containing cells, upon their transition into a post-symbiotic state.

Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease

Science.gov (United States)

Aldhous, Marian C.; Abu Bakar, Suhaili; Prescott, Natalie J.; Palla, Raquel; Soo, Kimberley; Mansfield, John C.; Mathew, Christopher G.; Satsangi, Jack; Armour, John A.L.

2010-01-01

The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case–control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case–control studies. PMID:20858604

Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease.

Science.gov (United States)

Aldhous, Marian C; Abu Bakar, Suhaili; Prescott, Natalie J; Palla, Raquel; Soo, Kimberley; Mansfield, John C; Mathew, Christopher G; Satsangi, Jack; Armour, John A L

2010-12-15

The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case-control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case-control studies.

Positive muscle protein net balance and differential regulation of atrogene expression after resistance exercise and milk protein supplementation

DEFF Research Database (Denmark)

Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

2014-01-01

Purpose Resistance exercise and amino acid availability are positive regulators of muscle protein net balance (NB). However, anabolic responses to resistance exercise and protein supplementation deserve further elucidation. The purpose was to compare intakes of whey, caseinate (both: 0.30 g/kg lean...... body mass), or a non-caloric control after heavy resistance exercise on protein turnover and mRNA expressions of forkhead homeobox type O (FOXO) isoforms, muscle RING finger 1 (MuRF1), and Atrogin1 in young healthy males. Methods Protein turnover was determined by stable isotope-labeled leucine...

The Yin and Yang of human beta defensins in health and disease

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Aaron eWeinberg

2012-10-01

Full Text Available Rapidly evolving research examining the extended role of human beta-defensins (hBDs in chemoattraction, innate immune-mediated response and promotion of angiogenesis suggest that the collective effects of hBDs extend well beyond their antimicrobial mechanism(s. Indeed, the numerous basic cellular functions associated with hBDs demonstrate that these peptides have dual impact on health, as they may be advantageous under certain conditions, but potentially detrimental in others. The consequences of these functions are reflected in the overexpression of hBDs in diseases, such as psoriasis, and recently the association of hBDs with pro-tumoral signaling. The mechanisms regulating hBD response in health and disease are still being elucidated. Clearly the spectrum of function now attributed to hBD regulation identifies these molecules as important cellular regulators, whose appropriate expression is critical for proper immune surveillance; i.e., expression of hBDs in proximity to areas of cellular dysregulation may inadvertently exacerbate disease progression. Understanding the mechanism(s that regulate contextual signaling of hBDs is an important area of concentration in our laboratories. Using a combination of immunologic, biochemical and molecular biologic approaches, we have identified signaling pathways associated with hBD promotion of immune homeostasis and have begun to dissect the inappropriate role that beta-defensins may assume in disease.

Defensin from the ornate sheep tick Dermacentor marginatus and its effect on Lyme borreliosis spirochetes

Czech Academy of Sciences Publication Activity Database

Chrudimská, Tereza; Čeřovský, Václav; Slaninová, Jiřina; Rego, Ryan O. M.; Grubhoffer, Libor

2014-01-01

Roč. 46, č. 2 (2014), s. 165-170 ISSN 0145-305X R&D Projects: GA ČR(CZ) GAP302/11/1901 Institutional support: RVO:60077344 ; RVO:61388963 Keywords : Tick * Dermacentor marginatus * Defensin * Borrelia afzelii * Antimicrobial activity * Peptide synthesis Subject RIV: EE - Microbiology, Virology; EE - Microbiology, Virology (UOCHB-X) Impact factor: 2.815, year: 2014

The Alpha-Defensin Immunoassay and Leukocyte Esterase Colorimetric Strip Test for the Diagnosis of Periprosthetic Infection

Science.gov (United States)

Wyatt, M.C.; Beswick, A.D.; Kunutsor, S.K.; Wilson, M.J.; Whitehouse, M.R.; Blom, A.W.

2016-01-01

Background: Synovial biomarkers have recently been adopted as diagnostic tools for periprosthetic joint infection (PJI), but their utility is uncertain. The purpose of this systematic review and meta-analysis was to synthesize the evidence on the accuracy of the alpha-defensin immunoassay and leukocyte esterase colorimetric strip test for the diagnosis of PJI compared with the Musculoskeletal Infection Society diagnostic criteria. Methods: We performed a systematic review to identify diagnostic technique studies evaluating the accuracy of alpha-defensin or leukocyte esterase in the diagnosis of PJI. MEDLINE and Embase on Ovid, ACM, ADS, arXiv, CERN DS (Conseil Européen pour la Recherche Nucléaire Document Server), CrossRef DOI (Digital Object Identifier), DBLP (Digital Bibliography & Library Project), Espacenet, Google Scholar, Gutenberg, HighWire, IEEE Xplore (Institute of Electrical and Electronics Engineers digital library), INSPIRE, JSTOR (Journal Storage), OAlster (Open Archives Initiative Protocol for Metadata Harvesting), Open Content, Pubget, PubMed, and Web of Science were searched for appropriate studies indexed from inception until May 30, 2015, along with unpublished or gray literature. The classification of studies and data extraction were performed independently by 2 reviewers. Data extraction permitted meta-analysis of sensitivity and specificity with construction of receiver operating characteristic curves for each test. Results: We included 11 eligible studies. The pooled diagnostic sensitivity and specificity of alpha-defensin (6 studies) for PJI were 1.00 (95% confidence interval [CI], 0.82 to 1.00) and 0.96 (95% CI, 0.89 to 0.99), respectively. The area under the curve (AUC) for alpha-defensin and PJI was 0.99 (95% CI, 0.98 to 1.00). The pooled diagnostic sensitivity and specificity of leukocyte esterase (5 studies) for PJI were 0.81 (95% CI, 0.49 to 0.95) and 0.97 (95% CI, 0.82 to 0.99), respectively. The AUC for leukocyte esterase and PJI

Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

Science.gov (United States)

Boutonnat, J; Bonnefoix, T; Mousseau, M; Seigneurin, D; Ronot, X

1998-01-01

Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2

NARCIS (Netherlands)

Hooijberg, J. H.; Broxterman, H. J.; Kool, M.; Assaraf, Y. G.; Peters, G. J.; Noordhuis, P.; Scheper, R. J.; Borst, P.; Pinedo, H. M.; Jansen, G.

1999-01-01

Transfection of multidrug resistance proteins (MRPs) MRP1 and MRP2 in human ovarian carcinoma 2008 cells conferred a marked level of resistance to short-term (1-4 h) exposure to the polyglutamatable antifolates methotrexate (MTX; 21-74-fold), ZD1694 (4-138-fold), and GW1843 (101-156-fold). Evidence

The defensin from avocado (Persea americana var. drymifolia) PaDef induces apoptosis in the human breast cancer cell line MCF-7.

Science.gov (United States)

Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Salgado-Garciglia, Rafael; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2016-08-01

Antimicrobial peptides (AMPs) are cytotoxic to cancer cells; however, mainly the effects of AMPs from animals have been evaluated. In this work, we assessed the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on the MCF-7 cancer cell line (a breast cancer cell line) and evaluated its mechanism of action. PaDef inhibited the viability of MCF-7 cells in a concentration-dependent manner, with an IC50=141.62μg/ml. The viability of normal peripheral blood mononuclear cells was unaffected by this AMP. Additionally, PaDef induced apoptosis in MCF-7 cells in a time-dependent manner, but did not affect the membrane potential or calcium flow. In addition, PaDef IC50 induced the expression of cytochrome c, Apaf-1, and the caspase 7 and 9 genes. Likewise, this defensin induced the loss of mitochondrial Δψm and increased the phosphorylation of MAPK p38, which may lead to MCF-7 apoptosis by the intrinsic pathway. This is the first report of an avocado defensin inducing intrinsic apoptosis in cancer cells, which suggests that it could be a potential therapeutic molecule in the treatment of cancer. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Comparison of the nodule vs. root transcriptome of the actinorhizal plant Datisca glomerata: actinorhizal nodules contain a specific class of defensins.

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Irina V Demina

Full Text Available Actinorhizal root nodule symbioses are very diverse, and the symbiosis of Datisca glomerata has previously been shown to have many unusual aspects. In order to gain molecular information on the infection mechanism, nodule development and nodule metabolism, we compared the transcriptomes of D. glomerata roots and nodules. Root and nodule libraries representing the 3'-ends of cDNAs were subjected to high-throughput parallel 454 sequencing. To identify the corresponding genes and to improve the assembly, Illumina sequencing of the nodule transcriptome was performed as well. The evaluation revealed 406 differentially regulated genes, 295 of which (72.7% could be assigned a function based on homology. Analysis of the nodule transcriptome showed that genes encoding components of the common symbiosis signaling pathway were present in nodules of D. glomerata, which in combination with the previously established function of SymRK in D. glomerata nodulation suggests that this pathway is also active in actinorhizal Cucurbitales. Furthermore, comparison of the D. glomerata nodule transcriptome with nodule transcriptomes from actinorhizal Fagales revealed a new subgroup of nodule-specific defensins that might play a role specific to actinorhizal symbioses. The D. glomerata members of this defensin subgroup contain an acidic C-terminal domain that was never found in plant defensins before.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages

DEFF Research Database (Denmark)

Jena, Prajna; Mohanty, Soumitra; Mohanty, Tirthankar

2012-01-01

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil...... and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule...... proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane...

Association studies of the copy-number variable ß-defensin cluster on 8p23.1 in adenocarcinoma and chronic pancreatitis

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Taudien Stefan

2012-11-01

Full Text Available Abstract Background Human ß-defensins are a family of antimicrobial peptides located at the mucosal surface. Both sequence multi-site variations (MSV and copy-number variants (CNV of the defensin-encoding genes are associated with increased risk for various diseases, including cancer and inflammatory conditions such as psoriasis and acute pancreatitis. In a case–control study, we investigated the association between MSV in DEFB104 as well as defensin gene (DEF cluster copy number (CN, and pancreatic ductal adenocarcinoma (PDAC and chronic pancreatitis (CP. Results Two groups of PDAC (N=70 and CP (N=60 patients were compared to matched healthy control groups CARLA1 (N=232 and CARLA2 (N=160, respectively. Four DEFB104 MSV were haplotyped by PCR, cloning and sequencing. DEF cluster CN was determined by multiplex ligation-dependent probe amplification. Neither the PDAC nor the CP cohorts show significant differences in the DEFB104 haplotype distribution compared to the respective control groups CARLA1 and CARLA2, respectively. The diploid DEF cluster CN exhibit a significantly different distribution between PDAC and CARLA1 (Fisher’s exact test P=0.027, but not between CP and CARLA2 (P=0.867. Conclusion Different DEF cluster b CN distribution between PDAC patients and healthy controls indicate a potential protective effect of higher CNs against the disease.

A dual mechanism involved in membrane and nucleic acid disruption of AvBD103b, a new avian defensin from the king penguin, against Salmonella enteritidis CVCC3377.

Science.gov (United States)

Teng, Da; Wang, Xiumin; Xi, Di; Mao, Ruoyu; Zhang, Yong; Guan, Qingfeng; Zhang, Jun; Wang, Jianhua

2014-10-01

The food-borne bacterial gastrointestinal infection is a serious public health threat. Defensins are evolutionarily conserved innate immune components with broad-spectrum antibacterial activity that do not easily induce resistance. AvBD103b, an avian defensin with potent activity against Salmonella enteritidis, was isolated from the stomach contents of the king penguin (Aptenodytes patagonicus). To elucidate further the antibacterial mechanism of AvBD103b, its effect on the S. enteritidis CVCC3377 cell membrane and intracellular DNA was researched. The cell surface hydrophobicity and a N-phenyl-1-naphthylamine uptake assay demonstrated that AvBD103b treatment increased the cell surface hydrophobicity and outer membrane permeability. Atomic absorption spectrometry, ultraviolet spectrophotometry, flow cytometry, and transmission electron microscopy (TEM) indicated that AvBD103b treatment can lead to the release of the cellular contents and cell death through damage of the membrane. DNA gel retardation and circular dichroism analysis demonstrated that AvBD103b interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that AvBD103b affected cellular functions, such as DNA synthesis. Our results confirmed that AvBD103b exerts its antibacterial activity by damaging the cell membrane and interfering with intracellular DNA, ultimately causing cell death, and suggested that AvBD103b may be a promising candidate as an alternative to antibiotics against S. enteritidis.

Antibacterial Activity of Four Human Beta-Defensins: HBD-19, HBD-23, HBD-27, and HBD-29

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David Camerini

2012-03-01

Full Text Available Human β-defensins (HBD are a family of small antimicrobial peptides that play important roles in the innate and adaptive immune defenses against microbial infection. In this study, we predicted the mature sequences and assessed the antibacterial properties of synthetic HBD-19, HBD-23, HBD-27, and HBD-29 against three species of clinically relevant bacteria: Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. We also examined the cytotoxicity of each β-defensin to human cells. HBD-19 exhibited modest antibacterial effects against E. coli and S. aureus but had little effect on the growth of P. aeruginosa. HBD-23 exhibited substantial antibacterial effects against all three bacterial species and was particularly potent against the Gram-negative species, E. coli and P. aeruginosa. HBD-27 exerted modest antibacterial activity only towards S. aureus while HBD-29 had modest antibacterial activity for E. coli and P. aeruginosa. HBD-23 and HBD-27 showed little or no toxicity to human peripheral blood mononuclear cells, while HBD-19 and HBD-29 decreased cell viability by 20% at 30 μg/mL.

Comparing the efficiency of Er,Cr:YSGG laser and diode laser on human β-defensin-1 and IL-1β levels during the treatment of generalized aggressive periodontitis and chronic periodontitis.

Science.gov (United States)

Ertugrul, Abdullah Seckin; Tekin, Yasin; Talmac, Ahmet Cemil

2017-11-01

The aim of this study is to determine the suitability of the Er,Cr:YSGG and 940 ± 15-nm diode laser for the treatment of generalized aggressive periodontitis and chronic periodontitis by measuring the levels of human β-defensin-1 and IL-1β. A total of 26 patients were included in this study. The study was designed as a "split-mouth" experiment. We performed scaling and root planing in the right maxillary quadrant, scaling and root planning + Er,Cr:YSGG laser in the left maxillary quadrant, scaling and root planning + 940 ± 15-nm diode laser in the left mandibular quadrant, and only scaling and root planing in the right mandibular quadrant. The presence of human β-defensin-1 and IL-1β was analyzed with an ELISA. When the baseline and post-treatment human β-defensin-1 levels and IL-1β levels of the study groups were evaluated, a decrease in human β-defensin-1 and IL-1β were observed in the quadrant where the Er,Cr:YSGG laser was applied in both the generalized aggressive periodontitis group and the chronic periodontitis group. The use of the Er,Cr:YSGG laser at non-surgical periodontal treatment decreased both IL-1β and human β-defensin-1 levels. It is likely that Er,Cr:YSGG laser is more suitable for the treatment of generalized aggressive periodontitis and chronic periodontitis.

Macrolide Resistance Mediated by a Bifidobacterium breve Membrane Protein

OpenAIRE

Margolles, Abelardo; Moreno, José Antonio; van Sinderen, Douwe; de los Reyes-Gavilán, Clara G.

2005-01-01

A gene coding for a hypothetical membrane protein from Bifidobacterium breve was expressed in Lactococcus lactis. Immunoblotting demonstrated that this protein is located in the membrane. Phenotypical changes in sensitivity towards 21 antibiotics were determined. The membrane protein-expressing cells showed higher levels of resistance to several macrolides.

Human Antimicrobial Peptides and Proteins

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Guangshun Wang

2014-05-01

Full Text Available As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32 can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized

Eosinophils from patients with type 1 diabetes mellitus express high level of myeloid alpha-defensins and myeloperoxidase

Czech Academy of Sciences Publication Activity Database

Neuwirth, Aleš; Dobeš, Jan; Oujezdská, Jana; Ballek, Ondřej; Benešová, Martina; Sumnik, Z.; Včeláková, J.; Koloušková, S.; Obermannová, B.; Kolář, Michal; Štechová, K.; Filipp, Dominik

2012-01-01

Roč. 273, č. 2 (2012), s. 158-163 ISSN 0008-8749 R&D Projects: GA MŠk 2B08066 Institutional research plan: CEZ:AV0Z50520514 Keywords : type 1 diabetes * alpha-defensin * myeloperoxidase * granulocyte * eosinophil Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.743, year: 2012

Association of β-defensin copy number and psoriasis in three cohorts of European origin.

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Stuart, Philip E; Hüffmeier, Ulrike; Nair, Rajan P; Palla, Raquel; Tejasvi, Trilokraj; Schalkwijk, Joost; Elder, James T; Reis, Andre; Armour, John A L

2012-10-01

A single previous study has demonstrated significant association of psoriasis with copy number of β-defensin genes, using DNA from psoriasis cases and controls from Nijmegen and Erlangen. In this study, we attempted to replicate that finding in larger new cohorts from Erlangen (N=2,017) and Michigan (N=5,412), using improved methods for β-defensin copy number determination based on the paralog ratio test, and enhanced methods of analysis and association testing implemented in the CNVtools resource. We demonstrate that the association with psoriasis found in the discovery sample is maintained after applying improved typing and analysis methods (P=5.5 × 10(-4), odds ratio (OR)=1.25). We also find that the association is replicated in 2,616 cases and 2,526 controls from Michigan, although at reduced significance (P=0.014), but not in new samples from Erlangen (1,396 cases and 621 controls, P=0.38). Meta-analysis across all cohorts suggests a nominally significant association (P=6.6 × 10(-3)/2 × 10(-4)) with an effect size (OR=1.081) much lower than found in the discovery study (OR=1.32). This reduced effect size and significance on replication is consistent with a genuine but weak association.

Association of β-defensin copy number and psoriasis in three cohorts of European origin

Science.gov (United States)

Stuart, Philip E; Hüffmeier, Ulrike; Nair, Rajan P; Palla, Raquel; Tejasvi, Trilokraj; Schalkwijk, Joost; Elder, James T; Reis, Andre; Armour, John AL

2012-01-01

A single previous study has demonstrated significant association of psoriasis with copy number of beta-defensin genes, using DNA from psoriasis cases and controls from Nijmegen and Erlangen. In this study we attempted to replicate that finding in larger new cohorts from Erlangen (N = 2017) and Michigan (N = 5412), using improved methods for beta-defensin copy number determination based on the paralog ratio test (PRT), and enhanced methods of analysis and association testing implemented in the CNVtools resource. We demonstrate that the association with psoriasis found in the discovery sample is maintained after applying improved typing and analysis methods (p = 5.5 × 10−4, OR = 1.25). We also find that the association is replicated in 2616 cases and 2526 controls from Michigan, although at reduced significance (p = 0.014), but not in new samples from Erlangen (1396 cases and 621 controls, p = 0.38). Meta-analysis across all cohorts suggests a nominally significant association (p = 6.6 × 10−3/2 × 10−4) with an effect size (OR = 1.081) much lower than found in the discovery study (OR = 1.32). This reduced effect size and significance on replication is consistent with a genuine but weak association. PMID:22739795

Protein resistance of surfaces modified with oligo(ethylene glycol) aryl diazonium derivatives.

Science.gov (United States)

Fairman, Callie; Ginges, Joshua Z; Lowe, Stuart B; Gooding, J Justin

2013-07-22

Anti-fouling surfaces are of great importance for reducing background interference in biosensor signals. Oligo(ethylene glycol) (OEG) moieties are commonly used to confer protein resistance on gold, silicon and carbon surfaces. Herein, we report the modification of surfaces using electrochemical deposition of OEG aryl diazonium salts. Using electrochemical and contact angle measurements, the ligand packing density is found to be loose, which supports the findings of the fluorescent protein labelling that aryl diazonium OEGs confer resistance to nonspecific adsorption of proteins albeit lower than alkane thiol-terminated OEGs. In addition to protein resistance, aryl diazonium attachment chemistry results in stable modification. In common with OEG species on gold electrodes, OEGs with distal hydroxyl moieties do confer superior protein resistance to those with a distal methoxy group. This is especially the case for longer derivatives where superior coiling of the OEG chains is possible. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of protein/essential amino acids and resistance training on skeletal muscle hypertrophy: A case for whey protein

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Stout Jeffrey R

2010-06-01

Full Text Available Abstract Regardless of age or gender, resistance training or provision of adequate amounts of dietary protein (PRO or essential amino acids (EAA can increase muscle protein synthesis (MPS in healthy adults. Combined PRO or EAA ingestion proximal to resistance training, however, can augment the post-exercise MPS response and has been shown to elicit a greater anabolic effect than exercise plus carbohydrate. Unfortunately, chronic/adaptive response data comparing the effects of different protein sources is limited. A growing body of evidence does, however, suggest that dairy PRO, and whey in particular may: 1 stimulate the greatest rise in MPS, 2 result in greater muscle cross-sectional area when combined with chronic resistance training, and 3 at least in younger individuals, enhance exercise recovery. Therefore, this review will focus on whey protein supplementation and its effects on skeletal muscle mass when combined with heavy resistance training.

Structural basis of protein oxidation resistance: a lysozyme study.

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Marion Girod

Full Text Available Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD simulations, we find the protein parts with increased root-mean-square deviation (RMSD to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation.

Transcriptional profiling avian beta-defensins in chicken oviduct epithelial cells before and after infection with Salmonella enterica serovar Enteritidis

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Bailey R Hartford

2009-07-01

Full Text Available Abstract Background Salmonella enterica serovar Enteritidis (SE colonizes the ovary and oviduct of chickens without causing overt clinical signs which can lead to SE-contamination of the content and membrane of shell-eggs as well as hatchery eggs. The organism utilizes the Salmonella Pathogenicity Island-2 encoded type III secretion system (T3SS-2 to promote persistence in the oviduct of laying hens. In this study, reverse transcriptase-polymerase chain reaction (RT-PCR was carried out to determine the expression profiles of 14 known avian beta defensins (AvBDs in primary chicken oviduct epithelial cells (COEC before and after infections with a wild type SE strain and T3SS mutant SE strains carrying an inactivated sipA or pipB gene. Results Based on the expression levels in uninfected COEC, AvBDs can be loosely grouped into three categories with AvBD4-5 and AvBD9-12 being constitutively expressed at high levels; AvBD1, AvBD3, and AvBD13-14 at moderate levels; and AvBD2 and AvBD6-8 at minimal levels. Infection with the wild type SE strain temporarily repressed certain highly expressed AvBDs and induced the expression of minimally expressed AvBDs. The pipB mutant, compared to the wild type strain, had reduced suppressive effect on the expression of highly expressed AvBDs. Moreover, the pipB mutant elicited significantly higher levels of the minimally expressed AvBDs than the wild type SE or the sipA mutant did. Conclusion Chicken oviduct epithelial cells express most of the known AvBD genes in response to SE infection. PipB, a T3SS-2 effector protein, plays a role in dampening the β-defensin arm of innate immunity during SE invasion of chicken oviduct epithelium.

Transcriptional profiling avian beta-defensins in chicken oviduct epithelial cells before and after infection with Salmonella enterica serovar Enteritidis.

Science.gov (United States)

Ebers, Katie L; Zhang, C Yan; Zhang, M Zhenyu; Bailey, R Hartford; Zhang, Shuping

2009-07-30

Salmonella enterica serovar Enteritidis (SE) colonizes the ovary and oviduct of chickens without causing overt clinical signs which can lead to SE-contamination of the content and membrane of shell-eggs as well as hatchery eggs. The organism utilizes the Salmonella Pathogenicity Island-2 encoded type III secretion system (T3SS-2) to promote persistence in the oviduct of laying hens. In this study, reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out to determine the expression profiles of 14 known avian beta defensins (AvBDs) in primary chicken oviduct epithelial cells (COEC) before and after infections with a wild type SE strain and T3SS mutant SE strains carrying an inactivated sipA or pipB gene. Based on the expression levels in uninfected COEC, AvBDs can be loosely grouped into three categories with AvBD4-5 and AvBD9-12 being constitutively expressed at high levels; AvBD1, AvBD3, and AvBD13-14 at moderate levels; and AvBD2 and AvBD6-8 at minimal levels. Infection with the wild type SE strain temporarily repressed certain highly expressed AvBDs and induced the expression of minimally expressed AvBDs. The pipB mutant, compared to the wild type strain, had reduced suppressive effect on the expression of highly expressed AvBDs. Moreover, the pipB mutant elicited significantly higher levels of the minimally expressed AvBDs than the wild type SE or the sipA mutant did. Chicken oviduct epithelial cells express most of the known AvBD genes in response to SE infection. PipB, a T3SS-2 effector protein, plays a role in dampening the beta-defensin arm of innate immunity during SE invasion of chicken oviduct epithelium.

Targeting protein kinases to reverse multidrug resistance in sarcoma.

Science.gov (United States)

Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

2016-02-01

Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of the breast cancer resistance protein in breast cancer

NARCIS (Netherlands)

Faneyte, Ian F.; Kristel, Petra M. P.; Maliepaard, Marc; Scheffer, George L.; Scheper, Rik J.; Schellens, Jan H. M.; van de Vijver, Marc J.

2002-01-01

PURPOSE: The breast cancer resistance protein (BCRP) is involved in in vitro multidrug resistance and was first identified in the breast cancer cell line MCF7/AdrVp. The aim of this study was to investigate the role of BCRP in resistance of breast cancer to anthracycline treatment. EXPERIMENTAL

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology.

Science.gov (United States)

Shen, Yang; Ai, Hong-Xin; Song, Ren; Liang, Zhen-Ning; Li, Jian-Feng; Zhang, Shuang-Quan

2010-10-20

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide. Copyright © 2010 Elsevier GmbH. All rights reserved.

Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

NARCIS (Netherlands)

Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

2005-01-01

The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC

Prokaryotic Expression of Reindeer β-defensin-1 (reBD-1) Gene and Bioactivity Identification of the Recombinant Protein%驯鹿β-防御素-1(reBD-1)基因的原核表达及蛋白活性的鉴定

Institute of Scientific and Technical Information of China (English)

苏丽娜; 杨银凤; 景岚; 曹贵方

2011-01-01

The aim of this experiment was to construct prokaryotic expression vector of reindeer β-defensin-l(reBD-l), induce the expression of reBD-1 fusion protein in E. Coli, and evaluate the bioactivity of the expression products. The prepro-peptide of reBD-1 was amplified by RT-PCR. The mature peptide of encoding reBD-1 was amplified from recombinant cloning vector PMD19T/ reBD-1, and then cloned into pET-32a (+ ), in which reBD-1 fusion protein expression was induced by IPTG in E. Coli BL2KDE3). The expressed product was further cultured and purified for experiment of bacteriostasis in vitro. The results showed that the amplified products of prepro-peptide and mature peptide were 215 and 138 bp, respectively, and the homology of the sequences of the targeted gene and reBD-1 mRNA was up to 100%. The molecular weight of fusion proteins of prepro-peptide and mature peptide were 28 and 24 ku. The agar diffusion method has demonstrated that 0. 08 mg · mL-1 purified mature peptide protein has obvious antimicrobial activity against S. Aureus and E. Coli. From the results we can conclude that the prepro-peptide and mature peptides have high expression level in E. Coli, and the mature peptide has resistance to both Gram negative and Gram positive bacteria.%旨在构建驯鹿β防御素-1(Reindeerβ-defensin-1,reBD-1)基因的原核表达载体pET-32a(+ )/reBD-1,诱导reBD-1融合蛋白在大肠杆菌中表达,并对其表达产物的生物学活性进行评价.利用RT-PCR技术扩增reBD-1前原肽.从重组克隆载体PMD19T/reBD-1中扩增reBD-1成熟肽编码基因,并克隆入pET-32a(+)中,在大肠杆菌BL21 (DE3)中用IPTG诱导表达reBD-1融合蛋白.表达的融合蛋白扩大培养,进一步纯化后进行体外抑菌试验.结果表明,reBD-1前原肽和成熟肽扩增产物大小分别为215和138 bp,目的基因的序列与驯鹿防御素-1 mRNA序列同源性为100％.前原肽和成熟肽融合蛋白分子量分别为28和24 ku.利用琼脂糖扩散法表明,0

Expression of natural antimicrobial peptide β-defensin-2 and Langerhans cell accumulation in epidermis from human non-healing leg ulcers

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Urszula Wojewodzka

2011-08-01

Full Text Available Chronic wounds like venous calf and diabetic foot ulcers are frequently contaminated and colonized by bacteria and it remains unclear whether there is sufficient expression of defensins and recruitment of epidermal Langerhans cells in the margin of ulcer compared to normal skin. The aim of this study was to examine immunohistochemically the expression of β-defensin-2 (hBD2, GM-CSF, VEGF growth factors and accumulation of CD1a+ Langerhans cells (LC in epidermis from chronic skin ulcers and to compare it to normal skin from the corresponding areas. Studies were carried out in 10 patients with diabetic foot, 10 patients with varicous ulcers of the calf and 10 patients undergoing orthopedic surgery (normal skin for control. Biopsy specimens were immunostained using specific primary antibodies, LSAB+ kit based on biotin-avidinperoxidase complex technique and DAB chromogen. Results were expressed as a mean staining intensity. Statistical analysis of staining showed significantly higher staining of hBD2 in both normal and ulcerated epidermis from foot sole skin compared to calf skin (normal and ulcerated, p<0.05. Chronic ulcers showed the same expression of hBD2 as normal skin. There was significantly lower accumulation of CD1a+ LC in normal epidermis from foot sole skin compared to normal calf skin (p<0.05. Accumulation of CD1a+ LC and GM-CSF upregulation at the border area of diabetic foot ulcer and reduction of LC concentration at the margin of venous calf ulcer compared to normal skin were observed. It seems that normal calf and sole epidermis is, unlike in the mechanisms of innate immunity, influenced by the different keratinocyte turnover and bacterial flora colonizing these regions. Insufficient upregulation of hBD2 in both diabetic foot and venous calf ulcers may suggest the pathological role of this protein in the chronicity of ulcers.

Antiprotozoan and Antiviral Activities of Non-Cytotoxic Truncated and Variant Analogues of Mussel Defensin

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Philippe Roch

2004-01-01

Full Text Available We previously reported the crucial role displayed by loop 3 of defensin isolated from the Mediterranean mussel, Mytilus galloprovincialis, in antibacterial and antifungal activities. We now investigated antiprotozoan and antiviral activities of some previously reported fragments B, D, E, P and Q. Two fragments (D and P efficiently killed Trypanosoma brucei (ID50 4–12 μM and Leishmania major (ID50 12–45 μM in a time/dose-dependent manner. Killing of T. brucei started as early as 1 h after initiation of contact with fragment D and reached 55% mortality after 6 h. Killing was temperature dependent and a temperature of 4°C efficiently impaired the ability to kill T. brucei. Fragments bound to the entire external epithelium of T. brucei. Prevention of HIV-1 infestation was obtained only with fragments P and Q at 20 μM. Even if fragment P was active on both targets, the specificity of fragments D and Q suggest that antiprotozoan and antiviral activities are mediated by different mechanisms. Truncated sequences of mussel defensin, including amino acid replacement to maintain 3D structure and increased positive net charge, also possess antiprotozoan and antiviral capabilities. New alternative and/or complementary antibiotics can be derived from the vast reservoir of natural antimicrobial peptides (AMPs contained in marine invertebrates.

Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan

NARCIS (Netherlands)

Jonker, JW; Smit, JW; Brinkhuis, RF; Maliepaard, M; Beijnen, JH; Schellens, JHM; Schinkel, AH

2000-01-01

Background and Methods: Breast cancer resistance protein (BCRP/MXR/ABCP) is a multidrug-resistance protein that is a member of the adenosine triphosphate-binding cassette family of drug transporters. BCRP can render tumor cells resistant to the anticancer drugs topotecan, mitoxantrone, doxorubicin,

The Effect of Calcipotriol on the Expression of Human β Defensin-2 and LL-37 in Cultured Human Keratinocytes

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Beom Joon Kim

2009-01-01

Full Text Available Background. Vitamin D has been reported to regulate innate immunity by controlling the expression of antimicrobial peptides (AMPs. Objective. We investigated the effect of calcipotriol on the expression of AMPs in human cultured keratinocytes. Methods. Keratinocytes were treated with lipopolysaccharide (LPS, TNF-α, Calcipotriol and irradiated with UVB, cultured, and harvested. To assess the expression of human beta defensin-2 and LL-37 in the control group, not exposed to any stimulants, the experimental group was treated with LPS, TNF-α, or UVB, and another group was treated again with calcipotriol; reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical staining were performed. Results. In the experimental group treated with LPS, UVB irradiation, and TNF-α, the expression of β-defensin and LL-37 was increased more than in the control group and then decreased in the experimental group treated with calcipotriol. Conclusions. Calcipotriol suppressed HBD-2 and LL-37, which were stimulated by UVB, LPS, and TNF-α.

Effect of yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of secretory immunoglobulin A and human beta-defensin 2 in healthy adult volunteers

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Kabeerdoss Jayakanthan

2011-12-01

Full Text Available Abstract Background Probiotics are used to provide health benefits. The present study tested the effect of a probiotic yoghurt on faecal output of beta-defensin and immunoglobulin A in a group of young healthy women eating a defined diet. Findings 26 women aged 18-21 (median 19 years residing in a hostel were given 200 ml normal yoghurt every day for a week, followed by probiotic yoghurt containing Bifidobacterium lactis Bb12® (109 in 200 ml for three weeks, followed again by normal yoghurt for four weeks. Stool samples were collected at 0, 4 and 8 weeks and assayed for immunoglobulin A and human beta-defensin-2 by ELISA. All participants tolerated both normal and probiotic yoghurt well. Human beta-defensin-2 levels in faeces were not altered during the course of the study. On the other hand, compared to the basal sample, faecal IgA increased during probiotic feeding (P = 0.0184 and returned to normal after cessation of probiotic yoghurt intake. Conclusions Bifidobacterium lactis Bb12® increased secretory IgA output in faeces. This property may explain the ability of probiotics to prevent gastrointestinal and lower respiratory tract infections.

Contemporary Issues in Protein Requirements and Consumption for Resistance Trained Athletes

Directory of Open Access Journals (Sweden)

Wilson Jacob

2006-06-01

Full Text Available Abstract In recent years an explosion of research papers concerning protein consumption has been published. The need to consolidate this information has become critical from both practical and future research standpoints. For this reason, the following paper presents an in depth analysis of contemporary issues in protein requirements and consumption for resistance trained athletes. Specifically, the paper covers: 1. protein requirements for resistance trained athletes; 2. the effect of the digestion rate of protein on muscular protein balance; 3. the optimal timing of protein intake relative to exercise; 4. the optimal pattern of protein ingestion, relative to how an individual should consume their protein throughout a 24 hour period, and what sources are utilized during this time frame; 5. protein composition and its interaction with measures of protein balance and strength performance; 6. the combination of protein and carbohydrates on plasma insulin levels and protein balance; 7. the efficacy of protein supplements and whole food protein sources. Our goal is to provide the reader with practical information in optimizing protein intake as well as for provision of sound advice to their clients. Finally, special care was taken to provide future research implications.

Identification of protein expression alterations in gefitinib-resistant human lung adenocarcinoma: PCNT and mPR play key roles in the development of gefitinib-associated resistance

Energy Technology Data Exchange (ETDEWEB)

Lin, Chi-Chen [Institute of Biomedical Science, National Chung-Hsing University, Taichung, Taiwan (China); Institute of Biomedical Science, and Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taiwan (China); Department of Medical Research and Education, Taichung Veterans General Hospital, Taichung, Taiwan (China); Division of Chest Medicine, Department of Internal Medicine, Changhua Christian Hospital, Changhua, Taiwan (China); Chen, Jing-Ting [Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan (China); Lin, Meng-Wei [Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan (China); Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan (China); Chan, Chia-Hao [Department of Obstetrics and Gynecology, Hsinchu Mackay Memorial Hospital, Hsinchu 30071, Taiwan (China); Wen, Yueh-Feng [Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan (China); Department of Internal Medicine, National Taiwan University Hospital Hsinchu Branch, Hsinchu, Taiwan (China); Wu, Shin-Bei [Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan (China); Chung, Ting-Wen [Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan (China); Lyu, Kevin W. [Lutheran Medical Center, Brooklyn, NY (United States); Global Scholars Program, St. George' s University/Northumbria University, Newcastle upon Tyne (United Kingdom); Chou, Hsiu-Chuan, E-mail: chouhc@mail.nhcue.edu.tw [Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan (China); and others

2015-11-01

Gefitinib is the first-line chemotherapeutic drug for treating non-small cell lung cancer (NSCLC), which comprises nearly 85% of all lung cancer cases worldwide. However, most patients eventually develop drug resistance after 12–18 months of treatment. Hence, investigating the drug resistance mechanism and resistance-associated biomarkers is necessary. Two lung adenocarcinoma cell lines, PC9 and gefitinib-resistant PC9/Gef, were established for examining resistance mechanisms and identifying potential therapeutic targets. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used for examining global protein expression changes between PC9 and PC9/Gef. The results revealed that 164 identified proteins were associated with the formation of gefitinib resistance in PC9 cells. Additional studies using RNA interference showed that progesterone receptor membrane component 1 and pericentrin proteins have major roles in gefitinib resistance. In conclusion, the proteomic approach enabled identifying of numerous proteins involved in gefitinib resistance. The results provide useful diagnostic markers and therapeutic candidates for treating gefitinib-resistant NSCLC. - Highlights: • 164 proteins associated with gefitinib resistance were identified through proteomic analysis. • In this study, a lung adenocarcinoma and its gefitinib resistant partner were established. • mPR and PCNT proteins have evidenced to play important roles in gefitinib resistance.

Identification of protein expression alterations in gefitinib-resistant human lung adenocarcinoma: PCNT and mPR play key roles in the development of gefitinib-associated resistance

International Nuclear Information System (INIS)

Lin, Chi-Chen; Chen, Jing-Ting; Lin, Meng-Wei; Chan, Chia-Hao; Wen, Yueh-Feng; Wu, Shin-Bei; Chung, Ting-Wen; Lyu, Kevin W.; Chou, Hsiu-Chuan

2015-01-01

Gefitinib is the first-line chemotherapeutic drug for treating non-small cell lung cancer (NSCLC), which comprises nearly 85% of all lung cancer cases worldwide. However, most patients eventually develop drug resistance after 12–18 months of treatment. Hence, investigating the drug resistance mechanism and resistance-associated biomarkers is necessary. Two lung adenocarcinoma cell lines, PC9 and gefitinib-resistant PC9/Gef, were established for examining resistance mechanisms and identifying potential therapeutic targets. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used for examining global protein expression changes between PC9 and PC9/Gef. The results revealed that 164 identified proteins were associated with the formation of gefitinib resistance in PC9 cells. Additional studies using RNA interference showed that progesterone receptor membrane component 1 and pericentrin proteins have major roles in gefitinib resistance. In conclusion, the proteomic approach enabled identifying of numerous proteins involved in gefitinib resistance. The results provide useful diagnostic markers and therapeutic candidates for treating gefitinib-resistant NSCLC. - Highlights: • 164 proteins associated with gefitinib resistance were identified through proteomic analysis. • In this study, a lung adenocarcinoma and its gefitinib resistant partner were established. • mPR and PCNT proteins have evidenced to play important roles in gefitinib resistance.

Protein source in a high-protein diet modulates reductions in insulin resistance and hepatic steatosis in fa/fa Zucker rats.

Science.gov (United States)

Wojcik, Jennifer L; Devassy, Jessay G; Wu, Yinghong; Zahradka, Peter; Taylor, Carla G; Aukema, Harold M

2016-01-01

High-protein diets are being promoted to reduce insulin resistance and hepatic steatosis in metabolic syndrome. Therefore, the effect of protein source in high-protein diets on reducing insulin resistance and hepatic steatosis was examined. Fa/fa Zucker rats were provided normal-protein (15% of energy) casein, high-protein (35% of energy) casein, high-protein soy, or high-protein mixed diets with animal and plant proteins. The high-protein mixed diet reduced area under the curve for insulin during glucose tolerance testing, fasting serum insulin and free fatty acid concentrations, homeostatic model assessment index, insulin to glucose ratio, and pancreatic islet cell area. The high-protein mixed and the high-protein soy diets reduced hepatic lipid concentrations, liver to body weight ratio, and hepatic steatosis rating. These improvements were observed despite no differences in body weight, feed intake, or adiposity among high-protein diet groups. The high-protein casein diet had minimal benefits. A high-protein mixed diet was the most effective for modulating reductions in insulin resistance and hepatic steatosis independent of weight loss, indicating that the source of protein within a high-protein diet is critical for the management of these metabolic syndrome parameters. © 2015 The Obesity Society.

Human β-defensin 2 may inhibit internalisation of bacillus Calmette-Guérin (BCG) in bladder cancer cells.

Science.gov (United States)

Kim, Jung Hoon; Kim, Soon-Ja; Lee, Kyung Mee; Chang, In Ho

2013-10-01

To investigate whether secretion of human β-defensin 2 (HBD-2) is induced by bacillus Calmette-Guérin (BCG) and to determine whether HBD-2 affects BCG internalisation in bladder cancer cells. Reverse transcription-polymerase chain reaction analysis was used to determine whether HBD-2 mRNA increases after incubation with BCG. HBD-2 proteins in 5637 and T24 human bladder cancer cell lines were assayed by enzyme-linked immunosorbent assay. The internalisation rate was evaluated by double immunofluorescence assay and confocal microscopy to test the optimal dose of HBD-2 for BCG internalisation. We also investigated the difference in internalisation rates and cell viability between recombinant HBD-2 protein, anti-HBD-2 antibody, and HBD-2 plus anti-HBD-2 antibody pretreatments. BCG induced HBD-2 mRNA expression and HBD-2 production dose and time-dependently in bladder cancer cells and affected BCG internalisation. Pretreatment with recombinant HBD-2 protein lowered internalisation of BCG dose-dependently. Moreover, anti-HBD-2 antibody prevented the effect of HBD-2 on BCG internalisation in bladder cancer cells. The internalisation rate of BCG pretreated with anti-HBD-2 antibody was higher than that in the control in 5637 (P internalisation rate in cells pretreated with anti-HBD-2 antibody plus recombinant HBD-2 protein was higher than that in the control in 5637 (P internalisation, which plays an important role during the initiation and propagation of the immunotherapeutic response in bladder cancer cells. © 2013 The Authors. BJU International © 2013 BJU International.

Human β-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells

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Tianying Bian

2017-01-01

Full Text Available The aim of this study was to investigate the role of human β-defensin 3 (hBD3 in the initiation stage of atherosclerosis with human umbilical vein endothelial cells (HUVECs triggered by tumor necrosis factor- (TNF- α. The effects of hBD3 on TNF-α-induced endothelial injury and inflammatory response were evaluated. Our data revealed that first, hBD3 reduced the production of interleukin-6 (IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1, and macrophage migration inhibitory factor (MIF in HUVECs in a dose-dependent manner. In addition, hBD3 significantly prevented intracellular reactive oxygen species (ROS production by HUVECs. Second, western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of intracellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 in TNF-α-induced HUVECs. As a result, hBD3 inhibited monocyte adhesion to TNF-α-treated endothelial cells. Additionally, hBD3 suppressed TNF-α-induced F-actin reorganization in HUVECs. Third, hBD3 markedly inhibited NF-κB activation by decreasing the phosphorylation of IKK-α/β, IκB, and p65 subunit within 30 min. Moreover, the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK in the mitogen-activated protein kinase (MAPK pathway were also inhibited by hBD3 in HUVECs. In conclusion, hBD3 exerts anti-inflammatory and antioxidative effects in endothelial cells in response to TNF-α by inhibiting NF-κB and MAPK signaling.

Increased Prevalence of Activated Protein C Resistance During ...

African Journals Online (AJOL)

Background: Acquired resistance to protein C in pregnancy has been established as one of the factors associated with ..... diabetes, sickle cell disease, smoking, anti-phospholipid syndrome inherited thrombophilia, and previous history of.

The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump

NARCIS (Netherlands)

Zaman, G. J.; Flens, M. J.; van Leusden, M. R.; de Haas, M.; Mülder, H. S.; Lankelma, J.; Pinedo, H. M.; Scheper, R. J.; Baas, F.; Broxterman, H. J.

1994-01-01

The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an

Membrane protein resistance of oligo(ethylene oxide) self-assembled monolayers.

Science.gov (United States)

Vaish, Amit; Vanderah, David J; Vierling, Ryan; Crawshaw, Fay; Gallagher, D Travis; Walker, Marlon L

2014-10-01

As part of an effort to develop biointerfaces for structure-function studies of integral membrane proteins (IMPs) a series of oligo(ethylene oxide) self-assembled monolayers (OEO-SAMs) were evaluated for their resistance to protein adsorption (RPA) of IMPs on Au and Pt. Spectroscopic ellipsometry (SE) was used to determine SAM thicknesses and compare the RPA of HS(CH2)3O(CH2CH2O)6CH3 (1), HS(CH2)3O(CH2CH2O)6H (2), [HS(CH2)3]2CHO(CH2CH2O)6CH3 (3) and [HS(CH2)3]2CHO(CH2CH2O)6H (4), assembled from water. For both substrates, SAM thicknesses for 1 to 4 were found to be comparable indicating SAMs with similar surface coverages and OEO chain order and packing densities. Fibrinogen (Fb), a soluble plasma protein, and rhodopsin (Rd), an integral membrane G-protein coupled receptor, adsorbed to the SAMs of 1, as expected from previous reports, but not to the hydroxy-terminated SAMs of 2 and 4. The methoxy-terminated SAMs of 3 were resistant to Fb but, surprisingly, not to Rd. The stark difference between the adsorption of Rd to the SAMs of 3 and 4 clearly indicate that a hydroxy-terminus of the OEO chain is essential for high RPA of IMPs. The similar thicknesses and high RPA of the SAMs of 2 and 4 show the conditions of protein resistance (screening the underlying substrate, packing densities, SAM order, and conformational mobility of the OEO chains) defined from previous studies on Au are applicable to Pt. In addition, the SAMs of 4, exhibiting the highest resistance to Fb and Rd, were placed in contact with undiluted fetal bovine serum for 2h. Low protein adsorption (≈12.4ng/cm(2)), obtained under these more challenging conditions, denote a high potential of the SAMs of 4 for various applications requiring the suppression of non-specific protein adsorption. Published by Elsevier B.V.

Proteomic analysis of polypeptides captured from blood during extracorporeal albumin dialysis in patients with cholestasis and resistant pruritus.

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Marina Gay

Full Text Available Albumin dialysis using the molecular adsorbent recirculating system (MARS is a new therapeutic approach for liver diseases. To gain insight into the mechanisms involved in albumin dialysis, we analyzed the peptides and proteins absorbed into the MARS strong anion exchange (SAX cartridges as a result of the treatment of patients with cholestasis and resistant pruritus. Proteins extracted from the SAX MARS cartridges after patient treatment were digested with two enzymes. The resulting peptides were analyzed by multidimensional liquid chromatography coupled to tandem mass spectrometry. We identified over 1,500 peptide sequences corresponding to 144 proteins. In addition to the proteins that are present in control albumin-derived samples, this collection includes 60 proteins that were specific to samples obtained after patient treatment. Five of these proteins (neutrophil defensin 1 [HNP-1], secreted Ly-6/uPAR-related protein 1 [SLURP1], serum amyloid A, fibrinogen alpha chain and pancreatic prohormone were confirmed to be removed by the dialysis procedure using targeted selected-reaction monitoring MS/MS. Furthermore, capture of HNP-1 and SLURP1 was also validated by Western blot. Interestingly, further analyses of SLURP1 in serum indicated that this protein was 3-fold higher in cholestatic patients than in controls. Proteins captured by MARS share certain structural and biological characteristics, and some of them have important biological functions. Therefore, their removal could be related either to therapeutic or possible adverse effects associated with albumin dialysis.

A subchronic feeding safety evaluation of transgenic milk containing human β-defensin 3 on reproductive system of C57BL/6J mouse.

Science.gov (United States)

Gao, Ming-Qing; Zhang, Ruiqi; Yang, Yange; Luo, Yuru; Jiang, Ming; Zhang, Yingli; Zhang, Yong; Qing, Suzhu

2018-05-01

Bovine mastitis is an infectious disease of the mammary gland which has been generally treated by antibiotic delivery. While the increasing drug-resistant bacteria and the high consumption of the antibiotic had become a noticeable concern. In a previous study, a mammary special vector expressing human β-defensin 3 (hBD3) was transfected into bovine fetal fibroblasts to produce mastitis-resistant bovine. This investigation focused on potential unintended effects of transgenic milk containing hBD3 produced by these mastitis-resistant bovine on the reproductive system of C57BL/6J mice. Mice were fed with diets containing transgenic milk or conventional milk, nutritionally balanced to an AIN93G diet for 90 days, and non-milk diet was selected as the negative group. The reproductive system was given special attention including reproductive organ/body ratios, necropsy and histopathology, serum sex hormone, sperm parameters, estrus cycle and the expression level of some specific genes which could indicate the development and function of reproductive system. No diet-related significant differences were observed among three groups in this 90-day feeding study. The results indicated that hBD3 milk does not appear to exert any effect on the reproductive system in C57BL/6J rats compared with conventional milk or the control diet. Copyright © 2018 Elsevier Ltd. All rights reserved.

Retinol-Binding Protein 4 and Insulin Resistance in Polycystic Ovary Syndrome

OpenAIRE

Hutchison, Samantha K.; Harrison, Cheryce; Stepto, Nigel; Meyer, Caroline; Teede, Helena J.

2008-01-01

OBJECTIVE?Polycystic ovary syndrome (PCOS) is an insulin-resistant state with insulin resistance being an established therapeutic target; however, measurement of insulin resistance remains challenging. We aimed to 1) determine serum retinol-binding protein 4 (RBP4) levels (purported to reflect insulin resistance) in women with PCOS and control subjects, 2) examine the relationship of RBP4 to conventional markers of insulin resistance, and 3) examine RBP4 changes with interventions modulating ...

The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action.

Science.gov (United States)

Chaili, Siyang; Cheung, Ambrose L; Bayer, Arnold S; Xiong, Yan Q; Waring, Alan J; Memmi, Guido; Donegan, Niles; Yang, Soo-Jin; Yeaman, Michael R

2016-02-01

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The β-defensin gallinacin-6 is expressed in the chicken digestive tract and has antimicrobial activity against food-borne pathogens

NARCIS (Netherlands)

van Dijk, A.; Veldhuizen, E.J.A.; Kalkhove, S.I.C.; Tjeerdsma-van Bokhoven, J.L.M.; Romijn, R.A.; Haagsman, H.P.

2007-01-01

Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of ß-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken

Whey protein hydrolysate augments tendon and muscle hypertrophy independent of resistance exercise contraction mode

DEFF Research Database (Denmark)

Farup, Jean; Rahbek, S K; Vendelbo, M H

2014-01-01

In a comparative study, we investigated the effects of maximal eccentric or concentric resistance training combined with whey protein or placebo on muscle and tendon hypertrophy. 22 subjects were allocated into either a high-leucine whey protein hydrolysate + carbohydrate group (WHD) or a carbohy......In a comparative study, we investigated the effects of maximal eccentric or concentric resistance training combined with whey protein or placebo on muscle and tendon hypertrophy. 22 subjects were allocated into either a high-leucine whey protein hydrolysate + carbohydrate group (WHD...... or contraction mode effects. In conclusion, high-leucine whey protein hydrolysate augments muscle and tendon hypertrophy following 12 weeks of resistance training – irrespective of contraction mode....

Stress proteins and phytohormones: their role in formation of plant resistance

International Nuclear Information System (INIS)

Kosakivska, I.V.

2005-01-01

Full text: Using the disc-electrophoresis methods, we have studied protein biosynthesis of different plants, including 11 species of Orchidaceae, some other tropical and subtropical plants, 9 different fruit plants, and 4 cultivars of Triticum aestivum L. under stresses factors such as high and low temperature, clinostating, radioactive irradiation and osmotic shock. Specific and unspecific reactions of plants protein system on stresses were found. De novo synthesis of 35 and 45 kD polypeptides were observed in total and mitochondrial proteins fractions after heat-shock and radioactive irradiation. This suggests that mitochondries participate in formation of plant resistance. Intensive synthesis of ABA revealed as the universal reaction of all studied plants on action of different kinds of stresses. Specific changes in balance of phytohormones were found under different stresses. We observed the correlation between endogenous ABA, IAA and cytokinin level and plant resistance. We also found the interaction between the process of biosynthesis of proteins and phytohormone balance, as well as their direct participation in formation of plant resistance. (author)

Identification of seed proteins associated with resistance to pre-harvested aflatoxin contamination in peanut (Arachis hypogaea L

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Li Ling

2010-11-01

Full Text Available Abstract Background Pre-harvest infection of peanuts by Aspergillus flavus and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. Some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. However, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. Therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism. Results The objective of this study was to identify resistance-associated proteins in response to A. flavus infection under drought stress using two-dimensional electrophoresis with mass spectrometry. To identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar (YJ-1 and a susceptible cultivar (Yueyou 7 under well-watered condition, drought stress, and A. flavus infection with drought stress. A total of 29 spots showed differential expression between resistant and susceptible cultivars in response to A. flavus attack under drought stress. Among these spots, 12 protein spots that consistently exhibited an altered expression were screened by Image Master 5.0 software and successfully identified by MALDI-TOF MS. Five protein spots, including Oso7g0179400, PII protein, CDK1, Oxalate oxidase, SAP domain-containing protein, were uniquely expressed in the resistant cultivar. Six protein spots including low molecular weight heat shock protein precursor, RIO kinase, L-ascorbate peroxidase, iso-Ara h3, 50 S ribosomal protein L22 and putative 30 S ribosomal S9 were significantly up-regulated in the resistant

Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

Science.gov (United States)

Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

2016-09-01

The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. Copyright © 2016 Elsevier B.V. All

Simple Coatings to Render Polystyrene Protein Resistant

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Marcelle Hecker

2018-02-01

Full Text Available Non-specific protein adsorption is detrimental to the performance of many biomedical devices. Polystyrene is a commonly used material in devices and thin films. Simple reliable surface modification of polystyrene to render it protein resistant is desired in particular for device fabrication and orthogonal functionalisation schemes. This report details modifications carried out on a polystyrene surface to prevent protein adsorption. The trialed surfaces included Pluronic F127 and PLL-g-PEG, adsorbed on polystyrene, using a polydopamine-assisted approach. Quartz crystal microbalance with dissipation (QCM-D results showed only short-term anti-fouling success of the polystyrene surface modified with F127, and the subsequent failure of the polydopamine intermediary layer in improving its stability. In stark contrast, QCM-D analysis proved the success of the polydopamine assisted PLL-g-PEG coating in preventing bovine serum albumin adsorption. This modified surface is equally as protein-rejecting after 24 h in buffer, and thus a promising simple coating for long term protein rejection of polystyrene.

Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study.

Science.gov (United States)

West, Daniel W D; Abou Sawan, Sidney; Mazzulla, Michael; Williamson, Eric; Moore, Daniel R

2017-07-11

No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h) and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD)) performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey) or an energy-matched placebo (CHO) immediately post-exercise (0 h), and again the following morning (~10 h of recovery). A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest). Participants ingested [ 15 N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO ( P = 0.064; effect size (ES) = 0.61, PRO vs. CHO) during overnight recovery. Over 24 h, net balance was enhanced in PRO ( P = 0.036) but not in CHO ( P = 0.84; ES = 0.69, PRO vs. CHO), which was mediated primarily by a reduction in protein breakdown (PRO protein supplementation improved MVC (ES = 0.76), REP (ES = 0.44), and peak power (ES = 0.55). In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of exercise performance after a strenuous bout of resistance exercise.

Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells.

Science.gov (United States)

Lajczak, Natalia K; Saint-Criq, Vinciane; O'Dwyer, Aoife M; Perino, Alessia; Adorini, Luciano; Schoonjans, Kristina; Keely, Stephen J

2017-09-01

Bile acids and epithelial-derived human β-defensins (HβDs) are known to be important factors in the regulation of colonic mucosal barrier function and inflammation. We hypothesized that bile acids regulate colonic HβD expression and aimed to test this by investigating the effects of deoxycholic acid (DCA) and ursodeoxycholic acid on the expression and release of HβD1 and HβD2 from colonic epithelial cells and mucosal tissues. DCA (10-150 µM) stimulated the release of both HβD1 and HβD2 from epithelial cell monolayers and human colonic mucosal tissue in vitro In contrast, ursodeoxycholic acid (50-200 µM) inhibited both basal and DCA-induced defensin release. Effects of DCA were mimicked by the Takeda GPCR 5 agonist, INT-777 (50 μM), but not by the farnesoid X receptor agonist, GW4064 (10 μM). INT-777 also stimulated colonic HβD1 and HβD2 release from wild-type, but not Takeda GPCR 5 -/- , mice. DCA stimulated phosphorylation of the p65 subunit of NF-κB, an effect that was attenuated by ursodeoxycholic acid, whereas an NF-κB inhibitor, BMS-345541 (25 μM), inhibited DCA-induced HβD2, but not HβD1, release. We conclude that bile acids can differentially regulate colonic epithelial HβD expression and secretion and discuss the implications of our findings for intestinal health and disease.-Lajczak, N. K., Saint-Criq, V., O'Dwyer, A. M., Perino, A., Adorini, L., Schoonjans, K., Keely, S. J. Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells. © FASEB.

The expression and significance of P-glycoprotein, lung resistance protein and multidrug resistance-associated protein in gastric cancer

Directory of Open Access Journals (Sweden)

Li Yan

2009-11-01

Full Text Available Abstract Background To detect the expression of multidrug resistance molecules P-glycoprotein (P-gp, Lung resistnce protein (LRP and Multidrug resistance-associated protein (MRP and analyze the relationship between them and the clinico-pathological features. Methods The expressions of P-gp, LRP and MRP in formalin-fixed paraffin-embedded tissue sections from 59 gastric cancer patients were determined by a labbelled Streptavidin-Peroxidase (SP immunohistochemical technique, and the results were analyzed in correlation with clinicopathological data. None of these patients received chemotherapy prior to surgery. Results The positive rates of P-gp, LRP, MRP were 86.4%, 84.7% and 27.1%, respectively. The difference between the positive rate of P-gp and MRP was significant statistically, as well as the difference between the expression of MRP and LRP. No significant difference was observed between P-gp and LRP, but the positively correlation between the expression of P-gp and LRP had been found. No significant correlation between the expression of P-gp, LRP, MRP and the grade of differentiation were observed. The expression of P-gp was correlated with clinical stages positively (r = 0.742, but the difference with the expression of P-gp in different stages was not significant. Conclusion The expressions of P-gp, LRP and MRP in patients with gastric cancer without prior chemotherapy are high, indicating that innate drug resistance may exist in gastric cancer.

Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter.

OpenAIRE

Neyfakh, A A; Borsch, C M; Kaatz, G W

1993-01-01

The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

Protein-protein association and cellular localization of four essential gene products encoded by tellurite resistance-conferring cluster "ter" from pathogenic Escherichia coli.

Science.gov (United States)

Valkovicova, Lenka; Vavrova, Silvia Minarikova; Mravec, Jozef; Grones, Jozef; Turna, Jan

2013-12-01

Gene cluster "ter" conferring high tellurite resistance has been identified in various pathogenic bacteria including Escherichia coli O157:H7. However, the precise mechanism as well as the molecular function of the respective gene products is unclear. Here we describe protein-protein association and localization analyses of four essential Ter proteins encoded by minimal resistance-conferring fragment (terBCDE) by means of recombinant expression. By using a two-plasmid complementation system we show that the overproduced single Ter proteins are not able to mediate tellurite resistance, but all Ter members play an irreplaceable role within the cluster. We identified several types of homotypic and heterotypic protein-protein associations among the Ter proteins by in vitro and in vivo pull-down assays and determined their cellular localization by cytosol/membrane fractionation. Our results strongly suggest that Ter proteins function involves their mutual association, which probably happens at the interface of the inner plasma membrane and the cytosol.

Role of urinary cathelicidin LL-37 and human β-defensin 1 in uncomplicated Escherichia coli urinary tract infections

DEFF Research Database (Denmark)

Nielsen, Karen L; Dynesen, Pia; Larsen, Preben

2014-01-01

Cathelicidin (LL-37) and human β-defensin 1 (hBD-1) are important components of the innate defense in the urinary tract. The aim of this study was to characterize whether these peptides are important for developing uncomplicated Escherichia coli urinary tract infections (UTIs). This was investiga......Cathelicidin (LL-37) and human β-defensin 1 (hBD-1) are important components of the innate defense in the urinary tract. The aim of this study was to characterize whether these peptides are important for developing uncomplicated Escherichia coli urinary tract infections (UTIs......). This was investigated by comparing urinary peptide levels of UTI patients during and after infection to those of controls, as well as characterizing the fecal flora of participants with respect to susceptibility to LL-37 and in vivo virulence. Forty-seven UTI patients and 50 controls who had never had a UTI were...... included. Participants were otherwise healthy, premenopausal, adult women. LL-37 MIC levels were compared for fecal E. coli clones from patients and controls and were also compared based on phylotypes (A, B1, B2, and D). In vivo virulence was investigated in the murine UTI model by use of selected fecal...

Insulin resistance and protein energy metabolism in patients with advanced chronic kidney disease.

Science.gov (United States)

Siew, Edward D; Ikizler, Talat Alp

2010-01-01

Insulin resistance (IR), the reciprocal of insulin sensitivity is a known complication of advanced chronic kidney disease (CKD) and is associated with a number of metabolic derangements. The complex metabolic abnormalities observed in CKD such as vitamin D deficiency, obesity, metabolic acidosis, inflammation, and accumulation of "uremic toxins" are believed to contribute to the etiology of IR and acquired defects in the insulin-receptor signaling pathway in this patient population. Only a few investigations have explored the validity of commonly used assessment methods in comparison to gold standard hyperinsulinemic hyperglycemic clamp technique in CKD patients. An important consequence of insulin resistance is its role in the pathogenesis of protein energy wasting, a state of metabolic derangement characterized by loss of somatic and visceral protein stores not entirely accounted for by inadequate nutrient intake. In the general population, insulin resistance has been associated with accelerated protein catabolism. Among end-stage renal disease (ESRD) patients, enhanced muscle protein breakdown has been observed in patients with Type II diabetes compared to ESRD patients without diabetes. In the absence of diabetes mellitus (DM) or severe obesity, insulin resistance is detectable in dialysis patients and strongly associated with increased muscle protein breakdown, primarily mediated by the ubiquitin-proteasome pathway. Recent epidemiological data indicate a survival advantage and better nutritional status in insulin-free Type II DM patients treated with insulin sensitizer thiazolidinediones. Given the high prevalence of protein energy wasting in ESRD and its unequivocal association with adverse clinical outcomes, insulin resistance may represent an important modifiable target for intervention in the ESRD population.

Insulin resistance enhances the mitogen-activated protein kinase signaling pathway in ovarian granulosa cells.

Directory of Open Access Journals (Sweden)

Linghui Kong

Full Text Available The ovary is the main regulator of female fertility. Granulosa cell dysfunction may be involved in various reproductive endocrine disorders. Here we investigated the effect of insulin resistance on the metabolism and function of ovarian granulosa cells, and dissected the functional status of the mitogen-activated protein kinase signaling pathway in these cells. Our data showed that dexamethasone-induced insulin resistance in mouse granulosa cells reduced insulin sensitivity, accompanied with an increase in phosphorylation of p44/42 mitogen-activated protein kinase. Furthermore, up-regulation of cytochrome P450 subfamily 17 and testosterone and down-regulation of progesterone were observed in insulin-resistant mouse granulosa cells. Inhibition of p44/42 mitogen-activated protein kinase after induction of insulin resistance in mouse granulosa cells decreased phosphorylation of p44/42 mitogen-activated protein kinase, downregulated cytochrome P450 subfamily 17 and lowered progesterone production. This insulin resistance cell model can successfully demonstrate certain mechanisms such as hyperandrogenism, which may inspire a new strategy for treating reproductive endocrine disorders by regulating cell signaling pathways.

Cross-resistance to purified Bt proteins, Bt corn and Bt cotton in a Cry2Ab2-corn resistant strain of Spodoptera frugiperda.

Science.gov (United States)

Yang, Fei; Kerns, David L; Head, Graham P; Price, Paula; Huang, Fangneng

2017-12-01

Gene-pyramiding by combining two or more dissimilar Bacillus thuringiensis (Bt) proteins into a crop has been used to delay insect resistance. The durability of gene-pyramiding can be reduced by cross-resistance. Fall armyworm, Spodoptera frugiperda, is a major target pest of the Cry2Ab2 protein used in pyramided Bt corn and cotton. Here, we provide the first experimental evaluation of cross-resistance in S. frugiperda selected with Cry2Ab2 corn to multiple Bt sources including purified Bt proteins, Bt corn and Bt cotton. Concentration - response bioassays showed that resistance ratios for Cry2Ab2-resistant (RR) relative to Cry2Ab2-susceptible (SS) S. frugiperda were -1.4 for Cry1F, 1.2 for Cry1A.105, >26.7 for Cry2Ab2, >10.0 for Cry2Ae and -1.1 for Vip3A. Larvae of Cry2Ab2-heterozygous (RS), SS and RR S. frugiperda were all susceptible to Bt corn and Bt cotton containing Cry1 (Cry1F or Cry1A.105) and/or Vip3A proteins. Pyramided Bt cotton containing Cry1Ac + Cry2Ab2 or Cry1Ab + Cry2Ae were also effective against SS and RS, but not RR. These findings suggest that Cry2Ab2-corn-selected S. frugiperda is not cross-resistant to Cry1F, Cry1A.105 or Vip3A protein, or corn and cotton plants containing these Bt proteins, but it can cause strong cross-resistance to Cry2Ae and Bt crops expressing similar Bt proteins. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

Science.gov (United States)

Sousa, Daniele O B; Carvalho, Ana F U; Oliveira, José Tadeu A; Farias, Davi F; Castelar, Ivan; Oliveira, Henrique P; Vasconcelos, Ilka M

2015-07-22

The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed.

Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi

Czech Academy of Sciences Publication Activity Database

Tonk, Miray; Cabezas-Cruz, A.; Valdés, James J.; Rego, Ryan O. M.; Chrudimská, Tereza; Strnad, Martin; Šíma, Radek; Bell-Sakyi, L.; Franta, Z.; Vilcinskas, A.; Grubhoffer, Libor; Rahnamaeian, M.

2014-01-01

Roč. 7, DEC 3 2015 (2014), s. 554 ISSN 1756-3305 R&D Projects: GA ČR(CZ) GAP302/11/1901; GA MŠk(CZ) EE2.3.30.0032; GA ČR GP13-12816P Institutional support: RVO:60077344 Keywords : Antimicrobial peptide * Defensin * Listeria grayi * Fusarium spp * Ixodes scapularis * Tick cell line Subject RIV: EE - Microbiology, Virology Impact factor: 3.430, year: 2014

Accurate measurement of gene copy number for human alpha-defensin DEFA1A3.

Science.gov (United States)

Khan, Fayeza F; Carpenter, Danielle; Mitchell, Laura; Mansouri, Omniah; Black, Holly A; Tyson, Jess; Armour, John A L

2013-10-20

Multi-allelic copy number variants include examples of extensive variation between individuals in the copy number of important genes, most notably genes involved in immune function. The definition of this variation, and analysis of its impact on function, has been hampered by the technical difficulty of large-scale but accurate typing of genomic copy number. The copy-variable alpha-defensin locus DEFA1A3 on human chromosome 8 commonly varies between 4 and 10 copies per diploid genome, and presents considerable challenges for accurate high-throughput typing. In this study, we developed two paralogue ratio tests and three allelic ratio measurements that, in combination, provide an accurate and scalable method for measurement of DEFA1A3 gene number. We combined information from different measurements in a maximum-likelihood framework which suggests that most samples can be assigned to an integer copy number with high confidence, and applied it to typing 589 unrelated European DNA samples. Typing the members of three-generation pedigrees provided further reassurance that correct integer copy numbers had been assigned. Our results have allowed us to discover that the SNP rs4300027 is strongly associated with DEFA1A3 gene copy number in European samples. We have developed an accurate and robust method for measurement of DEFA1A3 copy number. Interrogation of rs4300027 and associated SNPs in Genome-Wide Association Study SNP data provides no evidence that alpha-defensin copy number is a strong risk factor for phenotypes such as Crohn's disease, type I diabetes, HIV progression and multiple sclerosis.

Protein resistance of dextran and dextran-PEG copolymer films

Science.gov (United States)

Kozak, Darby; Chen, Annie; Bax, Jacinda; Trau, Matt

2011-01-01

The protein resistance of dextran and dextran-poly(ethylene glycol) (PEG) copolymer films was examined on an organosilica particle-based assay support. Comb-branched dextran-PEG copolymer films were synthesized in a two step process using the organosilica particle as a solid synthetic support. Particles modified with increasing amounts (0.1-1.2 mg m−2) of three molecular weights (10 000, 66 900, 400 000 g mol−1) of dextran were found to form relatively poor protein-resistant films compared to dextran-PEG copolymers and previously studied PEG films. The efficacy of the antifouling polymer films was found to be dependent on the grafted amount and its composition, with PEG layers being the most efficient, followed by dextran-PEG copolymers, and dextran alone being the least efficient. Immunoglobulin gamma (IgG) adsorption decreased from ~ 5 to 0.5 mg m−2 with increasing amounts of grafted dextran, but bovine serum albumin (BSA) adsorption increased above monolayer coverage (to ~2 mg m−2) indicating ternary adsorption of the smaller protein within the dextran layer. PMID:21614699

Protease-resistant prions selectively decrease Shadoo protein.

Directory of Open Access Journals (Sweden)

Joel C Watts

2011-11-01

Full Text Available The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C into PrP(Sc, a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho, a protein that resembles the flexibly disordered N-terminal domain of PrP(C, were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc. Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc. Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc during prion disease.

Dominant negative RPW8.2 fusion proteins reveal the importance of haustorium-oriented protein trafficking for resistance against powdery mildew in Arabidopsis.

Science.gov (United States)

Zhang, Qiong; Berkey, Robert; Pan, Zhiyong; Wang, Wenming; Zhang, Yi; Ma, Xianfeng; King, Harlan; Xiao, Shunyuan

2015-01-01

Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi.

The expression of cytokines and β -defensin 2, - 3, -4 in rabbit bone tissue after hydroxyapatite (HAp), α- Tricalcium phosphate (α-TCP) and polymethylmethacrylate (PMMA) implantation

International Nuclear Information System (INIS)

Vamze, J; Pilmane, M; Skagers, A

2012-01-01

Bone loss induced by inflammation is one of the complications after biomaterial implantation. There is no much data on expression of cytokines and defensins into the bone tissue around the implants in literature. The aim of this work was to investigate the distribution and appearance of interleukin (IL)-1, IL-6, IL-8, IL-10 and (β - defensin (BD)-2, BD-3, BD-4 after the implantation of different biomaterials. Bone developing zones, signs of bone-implant contact and low expression of pro-inflammatory cytokine IL-1, IL-6 and anti-inflammatory cytokine IL-10 in experimental tissue with pure HAp and unburned HAp implants indicate a potential advantage of this material in terms of its biocompatibility over the other materials used in our study.

1,25-Dihydroxyvitamin-D3 Induces Avian β-Defensin Gene Expression in Chickens.

Directory of Open Access Journals (Sweden)

Long Zhang

Full Text Available Host defense peptides (HDPs play a critical role in innate immunity. Specific modulation of endogenous HDP synthesis by dietary compounds has been regarded as a novel approach to boost immunity and disease resistance in animal production. 1,25-dihydroxy vitamin D3 (1,25D3 is well known as a powerful HDP inducer in humans, but limited information about the effect of 1,25D3 on HDPs in poultry is available. Here, we sought to examine whether 1,25D3 could stimulate avian β-defensin (AvBD expression in chickens. We used chicken embryo intestinal epithelial cells (CEIEPCs and peripheral blood mononuclear cells (PBMCs to study the effect of 1,25D3 on the expression of AvBDs. We observed that 1,25D3 is able to up-regulate the expression of several AvBDs in CEIEPCs and PBMCs, whereas it increased the amounts of AvBD4 mRNA in CEIEPCs only in the presence of lipopolysaccharide (LPS. On the other hand, LPS treatment not only inhibited the expression of CYP24A1 but also altered the expression pattern of VDR in CEIEPCs. Furthermore, AvBDs were not directly regulated by 1,25D3, as cycloheximide completely blocked 1,25D3-induced expression of AvBDs. Our observations suggest that 1,25D3 is capable of inducing AvBD gene expression and is a potential antibiotic alternative through augmentation of host innate immunity as well as disease control in chickens.

Role of multidrug resistance protein (MRP) in glutathione S-conjugate transport in mammalian cells

NARCIS (Netherlands)

Müller, M.; de Vries, E. G.; Jansen, P. L.

1996-01-01

The human multidrug resistance protein (MRP), a 190-kDa member of the ABC-protein superfamily, is an ATP-dependent glutathione S-conjugate carrier (GS-X pump) and is present in membranes of many, if not all, cells. Overexpression of MRP in tumor cells contributes to resistance to natural product

Role of multidrug resistance protein (MRP) in glutathione S-conjugate transport in mammalian cells

NARCIS (Netherlands)

Muller, M; deVries, EGE; Jansen, PLM

1996-01-01

The human multidrug resistance protein (MRP), a 190-kDa member of the ABC-protein superfamily, is an ATP-dependent glutathione S-conjugate carrier (GS-X pump) and is present in membranes of many, if not all, cells, Overexpression of MRP in tumor cells contributes to resistance to natural product

The heat-shock protein/chaperone network and multiple stress resistance.

Science.gov (United States)

Jacob, Pierre; Hirt, Heribert; Bendahmane, Abdelhafid

2017-04-01

Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multistress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat-shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone 'client proteins', many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat-shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signalling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/chaperone network. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Possibly similar genetic basis of resistance to Bacillus thuringiensis Cry1Ab protein in 3 resistant colonies of the sugarcane borer collected from Louisiana, USA.

Science.gov (United States)

Yang, Fei; Chen, Mao; Gowda, Anilkumar; Kerns, David L; Huang, Fangneng

2018-04-01

The sugarcane borer, Diatraea saccharalis (F.), is a major maize borer pest and a target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the mid-southern region of the United States. Evolution of resistance in target pest populations is a great threat to the long-term efficacy of Bt crops. In this study, we compared the genetic basis of resistance to Cry1Ab protein in 3 resistant colonies of sugarcane borer established from field populations in Louisiana, USA. Responses of larvae to the Cry1Ab protein for the parental and 10 other cross colonies were assayed in a diet-incorporated bioassay. All 3 resistant colonies were highly resistant to the Cry1Ab protein with a resistance ratio of >555.6 fold. No maternal effect or sex linkage was evident for the resistance in the 3 colonies; and the resistance was functionally nonrecessive at the Cry1Ab concentrations of ≤ 3.16 μg/g, but it became recessive at ≥10 μg/g. In an interstrain complementation test for allelism, the F 1 progeny from crosses between any 2 of the 3 resistant colonies exhibited the similar resistance levels as their parental colonies, indicating that the 3 colonies most likely shared a locus of Cry1Ab resistance. Results generated from this study should provide useful information in developing effective strategies for managing Bt resistance in the insect. © 2016 Institute of Zoology, Chinese Academy of Sciences.

The heat shock protein/chaperone network and multiple stress resistance

KAUST Repository

Jacob, Pierre; Hirt, Heribert; Bendahmane, Abdelhafid

2016-01-01

Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multi-stress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone

The heat shock protein/chaperone network and multiple stress resistance

KAUST Repository

Jacob, Pierre

2016-11-15

Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multi-stress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone

MRP proteins as potential mediators of heavy metal resistance in zebrafish cells.

Science.gov (United States)

Long, Yong; Li, Qing; Wang, Youhui; Cui, Zongbin

2011-04-01

Acquired resistance of mammalian cells to heavy metals is closely relevant to enhanced expression of several multidrug resistance-associated proteins (MRP), but it remains unclear whether MRP proteins confer resistance to heavy metals in zebrafish. In this study, we obtained zebrafish (Danio rerio) fibroblast-like ZF4 cells with resistance to toxic heavy metals after chronic cadmium exposure and selection for 6months. These cadmium-resistant cells (ZF4-Cd) were maintained in 5μM cadmium and displayed cross-resistance to cadmium, mercury, arsenite and arsenate. ZF4-Cd cells remained the resistance to heavy metals after protracted culture in cadmium-free medium. In comparison with ZF4-WT cells, ZF4-Cd cells exhibited accelerated rate of cadmium excretion, enhanced activity of MRP-like transport, elevated expression of abcc2, abcc4 and mt2 genes, and increased content of cellular GSH. Inhibition of MRP-like transport activity, GSH biosynthesis and GST activity significantly attenuated the resistance of ZF4-Cd cells to heavy metals. The results indicate that some of MRP transporters are involved in the efflux of heavy metals conjugated with cellular GSH and thus play crucial roles in heavy metal detoxification of zebrafish cells. Copyright © 2010 Elsevier Inc. All rights reserved.

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

OpenAIRE

Andreas Bayer; Justus Lammel; Mersedeh Tohidnezhad; Sebastian Lippross; Peter Behrendt; Tim Klüter; Thomas Pufe; Jochen Cremer; Holger Jahr; Franziska Rademacher; Regine Gläser; Jürgen Harder

2017-01-01

Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF?)) contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting acti...

Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervous system tumors.

Science.gov (United States)

Sasaki, Tsutomu; Hankins, Gerald R; Helm, Gregory A

2002-01-01

Lung resistance-related protein (LRP) was identified as the major vault protein (MVP), the main component of multimeric vault particles. It functions as a transport-associated protein that can be associated with multidrug resistance. In previous studies, expression of MVP/LRP has been documented in tumors of various types. In general, good correlations have been reported for expression of MVP/LRP and decreased sensitivity to chemotherapy and poor prognosis. MVP/LRP expression has been documented in glioblastomas, but its expression in nervous system tumors in general has not been well characterized. Immunohistochemistry using anti-human MVP/LRP antibody (LRP-56) was performed on formalin-fixed, paraffin-embedded archival tissue from 69 primary central nervous system tumors. Expression of MVP/LRP was observed in 81.2% (56/69) of primary nervous system tumors, including astrocytomas (11/13), oligodendrogliomas (1/2), oligoastrocytomas (5/5), ependymoma (1/1), meningiomas (35/45), schwannomas (2/2), and neurofibroma (1/1). Various degrees and distributions of immunoreactivity to MVP/ LRP were observed. Neither the presence nor the degree of immunoreactivity to MVP/LRP showed any correlation with either tumor grade or the presence of brain invasion.

Structure, function and subcellular localization of the potato Resistance protein Rx1

NARCIS (Netherlands)

Slootweg, E.J.

2009-01-01

Resistance proteins are part of the plant’s immune system and mediate a defence response upon recognizing their cognate pathogens. They are thought to be present in the cell as part of a larger protein complex. The modular architecture of R proteins suggests that they form a scaffold for various

Beta-defensins-2 expressions in gingival epithelium cells after probiotic Lactobacillus reuteri induction

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Tuti Kusumaningsih

2016-12-01

Full Text Available Background: Beta-defensins (BD are antimicrobial peptides that play a role in defense against pathogens. Beta-defensins (BD are expressed by a variety of epithelial cells, including gingival epithelium, salivary glands, saliva and salivary duct. BD-1 is expressed constitutively, while BD-2 and BD-3 expressions can be induced by commensal bacteria. Probiotics are commensal bacteria, thus L. reuteri as probiotic bacteria may act as “inducer” for BD-2 in epithelial gingiva. S. mutans is the main bacteria causing dental caries and sensitive to BD-2. Purpose: This study was aimed to prove that the administration of probiotic L. reuteri may improve BD-2 expressions in the gingiva epithelium. Method: This study was conducted in vivo using twenty-four male Rattus norvegicus Wistar strains aged 10-12 weeks and weighed 120-150 g. Those rats were randomly divided into four groups, namely negative control group (not induced with L. reuteri or S. mutans, positive control group (induced with S. mutans for 14 days, treatment group 1 (induced with L. reuteri for 14 days and S. mutans for 7 days, and treatment group 2 (induced with L. reuteri and S. mutans for 14 days concurrently. The concentration of L. reuteri used was 4x108cfu/ml, while the concentration of S. mutans was 1x 1010cfu/ml. 0.1 ml of each was dropped in the region of the mandibular incisors. BD-2 expression was calculated using immunohistochemical method. The difference of BD-2 expressions in gingival epithelial cells in the respective groups was analyzed by Anova/SPSS. Results: There were significant differences in BD-2 expressions in gingival epithelial cells in each group based on the results of Anova test (p=0.001. Conclusion: The administration of probiotic L. reuteri is able to increase BD-2 expressions in gingival epithelial cells.

Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study

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Daniel W. D. West

2017-07-01

Full Text Available No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey or an energy-matched placebo (CHO immediately post-exercise (0 h, and again the following morning (~10 h of recovery. A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest. Participants ingested [15N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO (P = 0.064; effect size (ES = 0.61, PRO vs. CHO during overnight recovery. Over 24 h, net balance was enhanced in PRO (P = 0.036 but not in CHO (P = 0.84; ES = 0.69, PRO vs. CHO, which was mediated primarily by a reduction in protein breakdown (PRO < CHO; P < 0.01. Exercise decreased repetitions to failure (REP, maximal strength (MVC, peak and mean power, and countermovement jump performance (CMJ at 0 h (all P < 0.05 vs. Pre. At 10 h, there were small-to-moderate effects for enhanced recovery of the MVC (ES = 0.56, mean power (ES = 0.49, and CMJ variables (ES: 0.27–0.49 in PRO. At 24 h, protein supplementation improved MVC (ES = 0.76, REP (ES = 0.44, and peak power (ES = 0.55. In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of

Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.

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Juliana Ide Aoki

2016-09-01

Full Text Available Tubercidin (TUB is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis.After transfection of a cosmid genomic library into L. major Friedlin (LmjF parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2 containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP. Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER, a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway.This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine

Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major.

Science.gov (United States)

Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

2016-09-01

Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected

The Alpha-Defensin Immunoassay and Leukocyte Esterase Colorimetric Strip Test for the Diagnosis of Periprosthetic Infection: A Systematic Review and Meta-Analysis.

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Wyatt, M C; Beswick, A D; Kunutsor, S K; Wilson, M J; Whitehouse, M R; Blom, A W

2016-06-15

Synovial biomarkers have recently been adopted as diagnostic tools for periprosthetic joint infection (PJI), but their utility is uncertain. The purpose of this systematic review and meta-analysis was to synthesize the evidence on the accuracy of the alpha-defensin immunoassay and leukocyte esterase colorimetric strip test for the diagnosis of PJI compared with the Musculoskeletal Infection Society diagnostic criteria. We performed a systematic review to identify diagnostic technique studies evaluating the accuracy of alpha-defensin or leukocyte esterase in the diagnosis of PJI. MEDLINE and Embase on Ovid, ACM, ADS, arXiv, CERN DS (Conseil Européen pour la Recherche Nucléaire Document Server), CrossRef DOI (Digital Object Identifier), DBLP (Digital Bibliography & Library Project), Espacenet, Google Scholar, Gutenberg, HighWire, IEEE Xplore (Institute of Electrical and Electronics Engineers digital library), INSPIRE, JSTOR (Journal Storage), OAlster (Open Archives Initiative Protocol for Metadata Harvesting), Open Content, Pubget, PubMed, and Web of Science were searched for appropriate studies indexed from inception until May 30, 2015, along with unpublished or gray literature. The classification of studies and data extraction were performed independently by 2 reviewers. Data extraction permitted meta-analysis of sensitivity and specificity with construction of receiver operating characteristic curves for each test. We included 11 eligible studies. The pooled diagnostic sensitivity and specificity of alpha-defensin (6 studies) for PJI were 1.00 (95% confidence interval [CI], 0.82 to 1.00) and 0.96 (95% CI, 0.89 to 0.99), respectively. The area under the curve (AUC) for alpha-defensin and PJI was 0.99 (95% CI, 0.98 to 1.00). The pooled diagnostic sensitivity and specificity of leukocyte esterase (5 studies) for PJI were 0.81 (95% CI, 0.49 to 0.95) and 0.97 (95% CI, 0.82 to 0.99), respectively. The AUC for leukocyte esterase and PJI was 0.97 (95% CI, 0.95 to 0

The increasing of beta-defensin-2 level in saliva after probiotic Lactobacillus reuteri administration

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Tuti Kusumaningsih

2015-03-01

Full Text Available Background: Commesal bacteria is an excellent inducer for beta defensin-2 (BD-2. Probiotics bacteria Lactobacillus reuteri (L. reuteri as commensal bacteria may play the same role as an excellent inducer for BD-2. Beta defensin is natural antimicrobial peptides widely expressed in oral cavity, including in epithelium salivary gland. Streptococcus mutans (S. mutans as the main of bacteria causing caries are sensitive to BD-2. Purpose: This research was aimed to determine whether administration of probiotic L. reuteri can increase salivary BD-2 level in Wistar rats. Methods: This research can be considered as a laboratory experimental research with a randomized control group post test only design. Twenty-four male Rattus norvegicus Wistar strain rats aged 3 months were used. They were randomly divided into four groups, namely two control groups (negative control group that was not induced and positive control group induced with S. mutans, and two treatment groups (K1: induced with L. reuteri for 14 days and S. mutans for 7 days, and K2: induced with L. reuteri and S. mutans simultaneously for 14 days. L. reuteri culture at a concentration of 108 CFU/ml and S. mutans culture at a concentration of 1010CFU/ml were induced into the oral cavity of Wistar rats. An examination of BD-2 level was then conducted by using Elisa techniques. results: There was significant difference of salivary BD-2 level among those treatment groups (p=0.001. BD-2 level in saliva was increased after the administration of L. reuteri. Conclusion: L. reuteri probiotic can increase salivary BD-2 level in Wistar rats.

Resistance Training and Co-supplementation with Creatine and Protein in Older Subjects with Frailty.

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Collins, J; Longhurst, G; Roschel, H; Gualano, B

2016-01-01

Studies assessing the effects co-supplementation with creatine and protein, along with resistance training, in older individuals with frailty are lacking. This is an exploratory trial from the Pro-Elderly study ("Protein Intake and Resistance Training in Aging") aimed at gathering knowledge on the feasibility, safety, and efficacy of co-supplementation with creatine and protein supplementation, combined with resistance training, in older individuals with frailty. A 14-week, double-blind, randomized, parallel-group, placebo controlled exploratory trial. The subjects were randomly assigned to whey protein and creatine co-supplementation (WHEY+CR) or whey protein supplementation (WHEY) group. All subjects undertook a supervised exercise training program and were assessed at baseline and after 14 weeks. Muscle function, body composition, blood parameters, and self-reported adverse events were assessed. No interaction effects (between-group differences) were observed for any dependent variables (p > 0.05 for all). However, there were main time-effects in handgrip (WHEY+CR = 26.65 ± 31.29; WHEY = 13.84 ± 14.93 Kg; p = 0.0005), timed-up-and-go (WHEY+CR = -11.20 ± 9.37; WHEY = -17.76 ± 21.74 sec; p = 0.006), and timed-stands test (WHEY+CR = 47.50 ± 35.54; WHEY = 46.87 ± 24.23 reps; p = 0.0001), suggesting that WHEY+CR and WHEY were similarly effective in improving muscle function. All of the subjects showed improvements in at least two of the three functional tests, regardless of their treatments. Body composition and blood parameters were not changed (p > 0.05). No severe adverse effects were observed. Co-supplementation with creatine and whey protein was well-tolerable and free of adverse events in older subjects with frailty undertaking resistance training. Creatine supplementation did not augment the adaptive effects of resistance training along with whey protein on body composition or muscle function in this population. Clinicaltrials.gov: NCT01890382.

Pea proteins oral supplementation promotes muscle thickness gains during resistance training: a double-blind, randomized, Placebo-controlled clinical trial vs. Whey protein

OpenAIRE

Babault, Nicolas; Pa?zis, Christos; Deley, Ga?lle; Gu?rin-Deremaux, Laetitia; Saniez, Marie-H?l?ne; Lefranc-Millot, Catherine; Allaert, Fran?ois A

2015-01-01

Background The effects of protein supplementation on muscle thickness and strength seem largely dependent on its composition. The current study aimed at comparing the impact of an oral supplementation with vegetable Pea protein (NUTRALYS?) vs. Whey protein and Placebo on biceps brachii muscle thickness and strength after a 12-week resistance training program. Methods One hundred and sixty one males, aged 18 to 35?years were enrolled in the study and underwent 12?weeks of resistance training o...

Skeletal muscle protein metabolism in the elderly: Interventions to counteract the 'anabolic resistance' of ageing

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Phillips Stuart M

2011-10-01

Full Text Available Abstract Age-related muscle wasting (sarcopenia is accompanied by a loss of strength which can compromise the functional abilities of the elderly. Muscle proteins are in a dynamic equilibrium between their respective rates of synthesis and breakdown. It has been suggested that age-related sarcopenia is due to: i elevated basal-fasted rates of muscle protein breakdown, ii a reduction in basal muscle protein synthesis (MPS, or iii a combination of the two factors. However, basal rates of muscle protein synthesis and breakdown are unchanged with advancing healthy age. Instead, it appears that the muscles of the elderly are resistant to normally robust anabolic stimuli such as amino acids and resistance exercise. Ageing muscle is less sensitive to lower doses of amino acids than the young and may require higher quantities of protein to acutely stimulate equivalent muscle protein synthesis above rest and accrue muscle proteins. With regard to dietary protein recommendations, emerging evidence suggests that the elderly may need to distribute protein intake evenly throughout the day, so as to promote an optimal per meal stimulation of MPS. The branched-chain amino acid leucine is thought to play a central role in mediating mRNA translation for MPS, and the elderly should ensure sufficient leucine is provided with dietary protein intake. With regards to physical activity, lower, than previously realized, intensity high-volume resistance exercise can stimulate a robust muscle protein synthetic response similar to traditional high-intensity low volume training, which may be beneficial for older adults. Resistance exercise combined with amino acid ingestion elicits the greatest anabolic response and may assist elderly in producing a 'youthful' muscle protein synthetic response provided sufficient protein is ingested following exercise.

Hypoxia-inducible factor-1α induces multidrug resistance protein in colon cancer

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Lv Y

2015-07-01

Full Text Available Yingqian Lv, Shan Zhao, Jinzhu Han, Likang Zheng, Zixin Yang, Li Zhao Department of Oncology, The Second Hospital, Hebei Medical University, Shijiazhuang, Hebei Province, People’s Republic of China Abstract: Multidrug resistance is the major cause of chemotherapy failure in many solid tumors, including colon cancer. Hypoxic environment is a feature for all solid tumors and is important for the development of tumor resistance to chemotherapy. Hypoxia-inducible factor (HIF-1α is the key transcription factor that mediates cellular response to hypoxia. HIF-1α has been shown to play an important role in tumor resistance; however, the mechanism is still not fully understood. Here, we found that HIF-1α and the drug resistance-associated gene multidrug resistance associated protein 1 (MRP1 were induced by treatment of colon cancer cells with the hypoxia-mimetic agent cobalt chloride. Inhibition of HIF-1α by RNA interference and dominant-negative protein can significantly reduce the induction of MRP1 by hypoxia. Bioinformatics analysis showed that a hypoxia response element is located at -378 to -373 bp upstream of the transcription start site of MRP1 gene. Luciferase reporter assay combined with mutation analysis confirmed that this element is essential for hypoxia-mediated activation of MRP gene. Furthermore, RNA interference revealed that HIF-1α is necessary for this hypoxia-driven activation of MRP1 promoter. Importantly, chromatin immunoprecipitation analysis demonstrated that HIF-1α could directly bind to this HRE site in vivo. Together, these data suggest that MRP1 is a downstream target gene of HIF-1α, which provides a potential novel mechanism for HIF-1α-mediated drug resistance in colon cancer and maybe other solid tumors as well. Keywords: hypoxia, hypoxia-inducible factor-1α, multidrug resistance associated protein, transcriptional regulation, chemotherapy tolerance

Resistance training with soy vs whey protein supplements in hyperlipidemic males

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Leddy John J

2009-03-01

Full Text Available Abstract Background Most individuals at risk for developing cardiovascular disease (CVD can reduce risk factors through diet and exercise before resorting to drug treatment. The effect of a combination of resistance training with vegetable-based (soy versus animal-based (whey protein supplementation on CVD risk reduction has received little study. The study's purpose was to examine the effects of 12 weeks of resistance exercise training with soy versus whey protein supplementation on strength gains, body composition and serum lipid changes in overweight, hyperlipidemic men. Methods Twenty-eight overweight, male subjects (BMI 25–30 with serum cholesterol >200 mg/dl were randomly divided into 3 groups (placebo (n = 9, and soy (n = 9 or whey (n = 10 supplementation and participated in supervised resistance training for 12 weeks. Supplements were provided in a double blind fashion. Results All 3 groups had significant gains in strength, averaging 47% in all major muscle groups and significant increases in fat free mass (2.6%, with no difference among groups. Percent body fat and waist-to-hip ratio decreased significantly in all 3 groups an average of 8% and 2%, respectively, with no difference among groups. Total serum cholesterol decreased significantly, again with no difference among groups. Conclusion Participation in a 12 week resistance exercise training program significantly increased strength and improved both body composition and serum cholesterol in overweight, hypercholesterolemic men with no added benefit from protein supplementation.

Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance

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Asunción Delgado

2017-05-01

Full Text Available Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance.

Dietary protein safety and resistance exercise: what do we really know?

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Lowery Lonnie M

2009-01-01

Full Text Available Abstract Resistance trainers continue to receive mixed messages about the safety of purposely seeking ample dietary protein in their quest for stimulating protein synthesis, improving performance, or maintaining health. Despite protein's lay popularity and the routinely high intakes exhibited by strength athletes, liberal and purposeful protein consumption is often maligned by "experts". University textbooks, instructors, and various forms of literature from personal training groups and athletic organizations continue to use dissuasive language surrounding dietary protein. Due to the widely known health benefits of dietary protein and a growing body of evidence on its safety profile, this is unfortunate. In response, researchers have critiqued unfounded educational messages. As a recent summarizing example, the International Society of Sports Nutrition (ISSN Position Stand: Protein and Exercise reviewed general literature on renal and bone health. The concluding remark that "Concerns that protein intake within this range [1.4 – 2.0 g/kg body weight per day] is unhealthy are unfounded in healthy, exercising individuals." was based largely upon data from non-athletes due to "a lack of scientific evidence". Future studies were deemed necessary. This assessment is not unique in the scientific literature. Investigators continue to cite controversy, debate, and the lack of direct evidence that allows it. This review discusses the few existing safety studies done specific to athletes and calls for protein research specific to resistance trainers. Population-specific, long term data will be necessary for effective education in dietetics textbooks and from sports governing bodies.

Identification of sociodemographic and clinical factors associated with the levels of human β-defensin-1 and human β-defensin-2 in the human milk of Han Chinese.

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Wang, Xiao-Fang; Cao, Rui-Ming; Li, Jing; Wu, Jing; Wu, Sheng-Mei; Chen, Tong-Xin

2014-03-14

Human milk provides infants with various immune molecules. The objective of the present study was to measure human β-defensin-1 (hBD-1) and human β-defensin-2 (hBD-2) levels in the colostrum and mature milk of healthy Han Chinese, to identify factors regulating milk hBD-1 and hBD-2 expression and to explore the potential protective effect of milk hBD-1 and hBD-2 on infants. A total of 100 mothers and their babies were recruited into the study. Sociodemographic characteristics and other factors were obtained by a questionnaire. Babies were followed up for a period of 6 months. Colostrum samples (n 100) and mature milk samples (n 82) were collected by hand expression. The hBD-1 and hBD-2 concentrations were measured by ELISA. The hBD-1 and hBD-2 levels differed in the colostrum and mature milk. In the colostrum, the concentration ranges of hBD-1 and hBD-2 were 1·04-12·81 μg/ml and 0·31-19·12 ng/ml, respectively. In mature milk, the hBD-1 and hBD-2 levels were 1·03-31·76 ng/ml and 52·65-182·29 pg/ml, respectively. Several independent factors influence their production. The multivariable analysis showed a strong association between pre-pregnancy BMI and hBD-1 levels in the colostrum (P=0·001), mode of delivery was significantly associated with hBD-2 levels in the colostrum (P=0·006) and gestational age was significantly associated with hBD-1 levels in mature milk (P= 0·010). During the first 6 months of life, the incidence rate of upper respiratory infection was found to be less in the high-colostrum hBD-1 group than in the low-colostrum hBD-1 group (χ²=4·995, P=0·025). The present study suggested that the abundance of hBD-1 in the colostrum may have a protective function against upper respiratory infection for infants younger than 6 months.

The role of organic proteins on the crack growth resistance of human enamel.

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Yahyazadehfar, Mobin; Arola, Dwayne

2015-06-01

With only 1% protein by weight, tooth enamel is the most highly mineralized tissue in mammals. The focus of this study was to evaluate contributions of the proteins on the fracture resistance of this unique structural material. Sections of enamel were obtained from the cusps of human molars and the crack growth resistance was quantified using a conventional fracture mechanics approach with complementary finite element analysis. In selected specimens the proteins were extracted using a potassium hydroxide treatment. Removal of the proteins resulted in approximately 40% decrease in the fracture toughness with respect to the fully proteinized control. The loss of organic content was most detrimental to the extrinsic toughening mechanisms, causing over 80% reduction in their contribution to the total energy to fracture. This degradation occurred by embrittlement of the unbroken bridging ligaments and consequent reduction in the crack closure stress. Although the organic content of tooth enamel is very small, it is essential to crack growth toughening by facilitating the formation of unbroken ligaments and in fortifying their potency. Replicating functions of the organic content will be critical to the successful development of bio-inspired materials that are designed for fracture resistance. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia

NARCIS (Netherlands)

de Vries, EGE; van Putten, WLJ; Verdonck, LF; Ossenkoppele, GJ; Verhoef, GEG; Vellenga, E

Despite treatment with intensive chemotherapy, a considerable number of patients with acute myeloid leukemia (AML) die from their disease due to the occurrence of resistance. Overexpression of the transporter proteins P-glycoprotein (P-gp) and multidrug resistance protein (MRP) 1 has been identified

Whey protein consumption after resistance exercise reduces energy intake at a post-exercise meal.

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Monteyne, Alistair; Martin, Alex; Jackson, Liam; Corrigan, Nick; Stringer, Ellen; Newey, Jack; Rumbold, Penny L S; Stevenson, Emma J; James, Lewis J

2018-03-01

Protein consumption after resistance exercise potentiates muscle protein synthesis, but its effects on subsequent appetite in this context are unknown. This study examined appetite and energy intake following consumption of protein- and carbohydrate-containing drinks after resistance exercise. After familiarisation, 15 resistance training males (age 21 ± 1 years, body mass 78.0 ± 11.9 kg, stature 1.78 ± 0.07 m) completed two randomised, double-blind trials, consisting of lower-body resistance exercise, followed by consumption of a whey protein (PRO 23.9 ± 3.6 g protein) or dextrose (CHO 26.5 ± 3.8 g carbohydrate) drink in the 5 min post-exercise. An ad libitum meal was served 60 min later, with subjective appetite measured throughout. Drinks were flavoured and matched for energy content and volume. The PRO drink provided 0.3 g/kg body mass protein. Ad libitum energy intake (PRO 3742 ± 994 kJ; CHO 4172 ± 1132 kJ; P = 0.007) and mean eating rate (PRO 339 ± 102 kJ/min; CHO 405 ± 154 kJ/min; P = 0.009) were lower during PRO. The change in eating rate was associated with the change in energy intake (R = 0.661, P = 0.007). No interaction effects were observed for subjective measures of appetite. The PRO drink was perceived as creamier and thicker, and less pleasant, sweet and refreshing (P consumption after resistance exercise reduces subsequent energy intake, and this might be partially mediated by a reduced eating rate. Whilst this reduced energy intake is unlikely to impair hypertrophy, it may be of value in supporting an energy deficit for weight loss.

FOLATE CYCLE GENE POLYMORPHISM AND ENDOGENOUS PEPTIDES IN CHILDREN WITH COW’S MILK PROTEIN ALLERGY

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T. A. Shumatova

2016-01-01

Full Text Available Folate cycle gene polymorphisms and the levels of endogenous antimicrobial peptides and proteins in the blood and coprofiltrates were studied in 45 children aged 3 to 12 months with cow’s milk protein allergy. The polymorphic variants of the MTHFR, MTRR, and MTR genes were shown to be considered as a risk factor for the development of allergy. There was a significant increase in the levels of zonulin, β-defensin 2, transthyretin, and eosinophil cationic protein in the coprofiltrates and in those of eotaxin, fatty acidbinding proteins, and membrane permeability-increasing protein in the serum (p<0.05. The finding can improve the diagnosis of the disease for a predictive purpose for the evaluation of the efficiency of performed therapy.

Babesial vector tick defensin against Babesia sp. parasites.

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Tsuji, Naotoshi; Battsetseg, Badgar; Boldbaatar, Damdinsuren; Miyoshi, Takeharu; Xuan, Xuenan; Oliver, James H; Fujisaki, Kozo

2007-07-01

Antimicrobial peptides are major components of host innate immunity, a well-conserved, evolutionarily ancient defensive mechanism. Infectious disease-bearing vector ticks are thought to possess specific defense molecules against the transmitted pathogens that have been acquired during their evolution. We found in the tick Haemaphysalis longicornis a novel parasiticidal peptide named longicin that may have evolved from a common ancestral peptide resembling spider and scorpion toxins. H. longicornis is the primary vector for Babesia sp. parasites in Japan. Longicin also displayed bactericidal and fungicidal properties that resemble those of defensin homologues from invertebrates and vertebrates. Longicin showed a remarkable ability to inhibit the proliferation of merozoites, an erythrocyte blood stage of equine Babesia equi, by killing the parasites. Longicin was localized at the surface of the Babesia sp. parasites, as demonstrated by confocal microscopic analysis. In an in vivo experiment, longicin induced significant reduction of parasitemia in animals infected with the zoonotic and murine B. microti. Moreover, RNA interference data demonstrated that endogenous longicin is able to directly kill the canine B. gibsoni, thus indicating that it may play a role in regulating the vectorial capacity in the vector tick H. longicornis. Theoretically, longicin may serve as a model for the development of chemotherapeutic compounds against tick-borne disease organisms.

Hepatitis B X-interacting protein promotes cisplatin resistance and regulates CD147 via Sp1 in ovarian cancer.

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Zou, Wei; Ma, Xiangdong; Yang, Hong; Hua, Wei; Chen, Biliang; Cai, Guoqing

2017-03-01

Ovarian cancer is the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure in malignant tumors. Hepatitis B X-interacting protein acts as an oncoprotein, regulates cell proliferation, and migration in breast cancer. We aimed to investigate the effects and mechanisms of hepatitis B X-interacting protein on resistance to cisplatin in human ovarian cancer cell lines. The mRNA and protein levels of hepatitis B X-interacting protein were detected using RT-PCR and Western blotting in cisplatin-resistant and cisplatin-sensitive tissues, cisplatin-resistant cell lines A2780/CP and SKOV3/CP, and cisplatin-sensitive cell lines A2780 and SKOV3. Cell viability and apoptosis were measured to evaluate cellular sensitivity to cisplatin in A2780/CP cells. Luciferase reporter gene assay was used to determine the relationship between hepatitis B X-interacting protein and CD147. The in vivo function of hepatitis B X-interacting protein on tumor burden was assessed in cisplatin-resistant xenograft models. The results showed that hepatitis B X-interacting protein was highly expressed in ovarian cancer of cisplatin-resistant tissues and cells. Notably, knockdown of hepatitis B X-interacting protein significantly reduced cell viability in A2780/CP compared with cisplatin treatment alone. Hepatitis B X-interacting protein and cisplatin cooperated to induce apoptosis and increase the expression of c-caspase 3 as well as the Bax/Bcl-2 ratio. We confirmed that hepatitis B X-interacting protein up-regulated CD147 at the protein expression and transcriptional levels. Moreover, we found that hepatitis B X-interacting protein was able to activate the CD147 promoter through Sp1. In vivo, depletion of hepatitis B X-interacting protein decreased the tumor volume and weight induced by cisplatin. Taken together, these results indicate that hepatitis B X-interacting protein promotes cisplatin resistance and regulated CD147 via Sp1 in

Co-induction of glucose regulated proteins and adriamycin resistance in Chinese hamster cells

International Nuclear Information System (INIS)

Shen, J.; Hughes, C.; Cai, J.; Bartels, C.; Gessner, T.; Subjeck, J.

1987-01-01

Glucose deprivation, anoxia, calcium ionophore A23187 or 2-deoxyglucose all inducers of glucose regulated proteins (grps), also lead to a significant induction of resistance to the drug adriamycin. In the case of anoxia, A23187 and 2-deoxyglucose, the induction of resistance correlates with both the application of the inducing stress and the induction of grps. In the case of glucose deprivation, the onset of resistance correlates with the onset of glucose deprivation and precedes grp induction. Removal of each grp including condition results in the rapid disappearance of this resistance in a manner which correlates with the repression of the grps. This drug resistance can be induced in confluent cells or in actively proliferating cells, although the effect is greater in the more sensitive proliferating cells. Induction of heat shock proteins (hsps) does not appear to lead to any major change in adriamycin resistance. Grp induced cells retain less adriamycin than do controls with the greatest reduction occurring during anoxia, which is also the strongest inducer of grps and resistance. The authors propose that the application of a grp inducing stress leads to a concurrent induction in drug resistance, possibly via the translocation of grps in the cell. Finally, they also observed that adriamycin itself can induce both hsps and grps. It is possible that adriamycin exposure may correspondingly induce auto-resistance

Pentapeptide-repeat proteins that act as topoisomerase poison resistance factors have a common dimer interface

International Nuclear Information System (INIS)

Vetting, Matthew W.; Hegde, Subray S.; Zhang, Yong; Blanchard, John S.

2011-01-01

The pentapeptide repeat protein AlbG, provides self-resistance to the nonribosomally encoded hybrid polyketide-peptide termed albicidin. Analysis of the AlbG three-dimensional structure and the sequences of other pentapeptide repeat proteins that confer resistance to topiosomerase poisons suggests they have a similar dimer interface which may be critical to their interaction with topoisomerases. The protein AlbG is a self-resistance factor against albicidin, a nonribosomally encoded hybrid polyketide-peptide with antibiotic and phytotoxic properties produced by Xanthomonas albilineans. Primary-sequence analysis indicates that AlbG is a member of the pentapeptide-repeat family of proteins (PRP). The structure of AlbG from X. albilineans was determined at 2.0 Å resolution by SAD phasing using data collected from a single trimethyllead acetate derivative on a home source. AlbG folds into a right-handed quadrilateral β-helix composed of approximately eight semi-regular coils. The regularity of the β-helix is blemished by a large loop/deviation in the β-helix between coils 4 and 5. The C-terminus of the β-helix is capped by a dimerization module, yielding a dimer with a 110 Å semi-collinear β-helical axis. This method of dimer formation appears to be common to all PRP proteins that confer resistance to topoisomerase poisons and contrasts with most PRP proteins, which are typically monomeric

Colistin resistance associated with outer membrane protein change in Klebsiella pneumoniae and Enterobacter asburiae.

Science.gov (United States)

Kádár, Béla; Kocsis, Béla; Tóth, Ákos; Kristóf, Katalin; Felső, Péter; Kocsis, Béla; Böddi, Katalin; Szabó, Dóra

2017-06-01

In this study, outer membrane proteins (OMPs) of colistin-resistant Klebsiella pneumoniae and Enterobacter asburiae were analyzed. One colistin-susceptible and three colistin-resistant K. pneumoniae sequence type 258 strains as well as one colistin-susceptible E. asburiae and its colistin-heteroresistant counterpart strain were involved in the study. OMP analysis of each strain was performed by microchip method. Matrix-assisted laser desorption ionization time of flight/mass spectrometry (MALDI-TOF/MS) investigation was carried out after separation of OMPs by two-dimensional gel electrophoresis and in-gel digestion. The MALDI-TOF/MS analysis of OMPs in the colistin-susceptible K. pneumoniae found 16 kDa proteins belonging to the LysM domain/BON superfamily, as well as DNA starvation proteins, whereas OmpX and OmpW were detected in the colistin-resistant counterpart strains. OmpC and OmpW were detected in the colistin-susceptible E. asburiae, whereas OmpA and OmpX were identified in the colistin-resistant counterpart. This study demonstrated that OMP differences were between colistin-susceptible and -resistant counterpart strains. The altered Gram-negative cell wall may contribute to acquired colistin resistance in Enterobacteriaceae.

Selection and characterization of resistance to the Vip3Aa20 protein from Bacillus thuringiensis in Spodoptera frugiperda.

Science.gov (United States)

Bernardi, Oderlei; Bernardi, Daniel; Horikoshi, Renato J; Okuma, Daniela M; Miraldo, Leonardo L; Fatoretto, Julio; Medeiros, Fernanda Cl; Burd, Tony; Omoto, Celso

2016-09-01

Spodoptera frugiperda is one the main target pests of maize events expressing Vip3Aa20 protein from Bacillus thuringiensis (Bt) in Brazil. In this study, we selected a resistant strain of S. frugiperda on Bt maize expressing Vip3Aa20 protein and characterized the inheritance and fitness costs of the resistance. The resistance ratio of the Vip3Aa20-resistant strain of S. frugiperda was >3200-fold. Neonates of the Vip3Aa20-resistant strain were able to survive and emerge as fertile adults on Vip3Aa20 maize, while larvae from susceptible and heterozygous strains did not survive. The inheritance of Vip3Aa20 resistance was autosomal recessive and monogenic. Life history studies to investigate fitness cost revealed an 11% reduction in the survival rate until adult stage and a ∼50% lower reproductive rate of the Vip3Aa20-resistant strain compared with susceptible and heterozygous strains. This is the first characterization of S. frugiperda resistance to Vip3Aa protein. Our results provide useful information for resistance management programs designed to prevent or delay resistance evolution to Vip3Aa proteins in S. frugiperda. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Salivary human beta defensins affected by oral Candida status in Chinese HIV/AIDS patients undergoing ART.

Science.gov (United States)

Liu, Zhenmin; Yong, Xiangzhi; Jiang, Lanlan; Zhang, Linlin; Lin, Xuefang; Liu, Wei; Peng, Yuanyuan; Tao, Renchuan

2018-03-02

To observe relationships between oral Candida status and salivary human beta defensin-2 and -3 (hBD-2 and hBD-3) levels in HIV/AIDS patients of Guangxi, China during the first year of antiretroviral therapy (ART) dynamically, and to understand the influence of ART on oral Candida status and salivary hBDs expressions. A prospective self-controlled study was carried to observe the dynamic changes of CD4 + T cell counts, oral Candida carriages and salivary hBD-2,3 expressions in HIV/AIDS patients during the first year of ART. A total of 90 HIV/AIDS patients were enrolled, and were examined at the baseline, 3rd, 6th, 12th month of ART. Thirty healthy individuals were enrolled as control. Peripheral blood, oral rinse sample and unstimulated whole saliva were collected to test CD4 + T cell counts, oral Candida carriages and hBD-2,3 expressions. In the first year of ART, CD4 + T cell counts increased significantly. However, oral Candida carriages and oral candidiasis decreased significantly, and salivary hBD-2 expressions in HIV/AIDS patients decreased gradually, salivary hBD-3 levels were highly variable. Salivary hBD-2 concentrations were positively related to oral Candida carriages. The incidence of oral candidiasis among HIV/AIDS patients gradually decreased due to the immune reconstruction of ART. Salivary defensins might play an important role in Candida-host interaction in HIV/AIDS patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Multi-Center, Double-Blind, Vehicle-Controlled Clinical Trial of an Alpha and Beta Defensin-Containing Anti-Aging Skin Care Regimen With Clinical, Histopathologic, Immunohistochemical, Photographic, and Ultrasound Evaluation.

Science.gov (United States)

Taub, Amy; Bucay, Vivian; Keller, Gregory; Williams, Jay; Mehregan, Darius

2018-04-01

Anti-aging strategies utilizing stem cells are in the forefront. Alpha and beta defensins are natural immune peptides that have been shown to activate an LGR6-positive stem cell locus in the hair follicle, identified as the source of most new epidermal cells during acute wound healing. We investigated the ability of biomimetic alpha and beta defensin molecules, supplemented with supportive cosmetic ingredients, formulated into three skin care products, at improving the structure and function of aging skin. A participant- and investigator -blinded, placebo-controlled, multi-center trial was performed in outpatient settings. Forty-four healthy female subjects, aged 41-71 years, skin types I-V, completed the study with 2/3 receiving full formula and 1/3 receiving the placebo formula. A skin care regimen of 3 products (serum, cream, and mask) containing alpha-defensin 5 and beta-defensin 3, and other cosmetic ingredients, was applied to the face, post-auricular, and neck skin two times per day for 12 weeks in those receiving full formula, whereas the placebo group received the identically packaged regimen without the active ingredients. Methods of evaluation included histopathology and immunohistochemistry (7 subjects), clinical evaluation of pores, superficial and deep wrinkles based on Griffiths scale, and high-resolution photography (all subjects). In addition, a subset of 15 patients were evaluated with the QuantifiCare system (3-dimensional imaging and skin care scores for evenness, pores, oiliness) and Cortex measurements (high-resolution skin ultrasound, TEWL, elasticity, color, and hydration). Data points for evaluation included baseline, 6 weeks, and 12 weeks. All patients used the same sunscreen and cleanser, which was provided to them. The full formula regimen caused a significantly (P equals 0.027) increased thickness of the epidermis as seen in histology, not seen in the placebo group, with no signs of inflammation. No excessive cell proliferation was

Testing of disease-resistance of pokeweed antiviral protein gene ...

African Journals Online (AJOL)

Transformation of pokeweed antiviral protein gene (PAP) into plants was shown to improve plant resistance to several viruses or fungi pathogens with no much negative effect on plant growth. The non-virulent defective PAP inhibits only the virus but does not interfere with the host. A non-virulent defective PAP gene ...

Effects of resistance training and protein supplementation on bone turnover in young adult women

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Sinning Wayne E

2005-08-01

Full Text Available Abstract Background The strength of aging bone depends on the balance between the resorption and formation phases of the remodeling process. The purpose of this study was to examine the interaction of two factors with the potential to exert opposing influences on bone turnover, resistance exercise training and high dietary protein intake. It was hypothesized that resistance training by young, healthy, untrained women with protein intakes near recommended levels (0.8 g·kg-1·d-1 would promote bone formation and/or inhibit bone resorption, and that subsequent supplementation to provide 2.4 g protein·kg-1·d-1 would reverse these effects. Methods Bone formation was assessed with serum bone-specific alkaline phosphatase (BAP and osteocalcin (OC, and bone resorption with urinary calcium and deoxypyridinoline (DPD. Biochemical, strength, anthropometric, dietary, and physical activity data were obtained from 24 healthy, untrained, eumenorrheic women (18–29y at baseline, after eight weeks of resistance training (3 d·wk-1, ~1 hr·d-1; 3 sets, 6–10 repetitions, 13 exercises, 75–85% maximum voluntary contraction, and after 12 weeks of resistance training and 10 days of protein/placebo supplementation. Subjects were randomized (double-blind to either a high protein (HP or training control (TC group and, during the final 10 days, consumed either enough purified whey protein to bring daily protein intake to 2.4 g·kg-1·d-1, or an equivalent dose of isoenergetic, carbohydrate placebo. Results Strength, lean tissue mass, and DPD increased significantly in both groups over time, while percent body fat and BAP decreased (repeated measures ANOVA, p ≤ 0.05, Bonferroni correction. No significant changes were observed for serum OC or urinary calcium, and no significant group (TC, HP × time (baseline, week 8, week 12 interactions emerged for any of the biochemical measures. Conclusion (1 Twelve weeks of high-intensity resistance training did not appear to

Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

Science.gov (United States)

Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

2013-01-01

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×103 and 95.4×103 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×105 and 622.2×105 cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals. PMID:23799010

The Role of ABC Proteins in Drug Resistant Breast Cancer Cells

Science.gov (United States)

2008-04-01

called the Plasmodium falciparum Chloroquine Transporter (PfCRT). While PfCRT is known to be the main molecular determinant of chloroquine resistance...proteins (such as human P-glycoprotein) and labeled PfCRT with a photoaffinity drug analogue . A manuscript is currently in preparation detailing my results...directly responsible for drug response, the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) (Fidock et al 2000). While not a member of

Mutation in ribosomal protein S5 leads to spectinomycin resistance in Neisseria gonorrhoeae.

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Elena eIlina

2013-07-01

Full Text Available Spectinomycin remains a useful reserve option for therapy of gonorrhea. The emergence of multidrug-resistant Neisseria gonorrhoeae strains with decreased susceptibility to cefixime and to ceftriaxone makes it the only medicine still effective for treatment of gonorrhea infection in analogous cases. However, adoption of spectinomycin as a routinely used drug of choice was soon followed by reports of spectinomycin resistance. The main molecular mechanism of spectinomycin resistance in N. gonorrhoeae was C1192T substitution in 16S rRNA genes. Here we reported a Thr-24→Pro mutation in ribosomal protein S5 found in spectinomycin resistant clinical N. gonorrhoeae strain, which carried no changes in 16S rRNA. In a series of experiments, the transfer of rpsE gene allele encoding the mutant ribosomal protein S5 to the recipient N. gonorrhoeae strains was analyzed. The relatively high rate of transformation (ca. 10-5 CFUs indicates the possibility of spread of spectinonycin resistance within gonococcal population due to the horizontal gene transfer.

Bactobolin resistance is conferred by mutations in the L2 ribosomal protein.

Science.gov (United States)

Chandler, Josephine R; Truong, Thao T; Silva, Patricia M; Seyedsayamdost, Mohammad R; Carr, Gavin; Radey, Matthew; Jacobs, Michael A; Sims, Elizabeth H; Clardy, Jon; Greenberg, E Peter

2012-12-18

Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). Currently available antibiotics target surprisingly few cellular functions, and there is a need to identify novel antibiotic targets. We have been interested in the Burkholderia thailandensis bactobolins, and we sought to learn about the target of bactobolin activity by mapping spontaneous resistance mutations in the bactobolin-sensitive Bacillus subtilis. Our results indicate that the bactobolin target is the 50S ribosome-associated L2 protein or a region of the ribosome affected by L2. Bactobolin-resistant mutants are not resistant to other known ribosome inhibitors. Our evidence indicates that bactobolins

Coat protein-mediated resistance against an Indian isolate of the ...

Indian Academy of Sciences (India)

Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were ...

High levels of X-linked Inhibitor-of-Apoptosis Protein (XIAP) are indicative of radio chemotherapy resistance in rectal cancer

International Nuclear Information System (INIS)

Flanagan, L.; Kehoe, J.; Fay, J.; Bacon, O.; Lindner, A.U.; Kay, E.W.; Deasy, J.; McNamara, D.A.; Prehn, J.H.M.

2015-01-01

The mainstay of treatment in rectal cancer is neoadjuvant radio chemotherapy prior to surgery, in an attempt to downstage the tumour, allowing for more complete removal during surgery. In 40 % of cases however, this neoadjuvant radio chemotherapy fails to achieve tumour regression, partly due insufficient apoptosis signaling. X-linked Inhibitor of Apoptosis Protein (XIAP) is an anti-apoptotic protein that has been reported to contribute to disease progression and chemotherapy resistance. We obtained rectal biopsy normal and matched tumour tissue from 29 rectal cancer patients with varying degrees of tumour regression, and using Western blot, examined anti-apoptotic XIAP and pro-apoptotic Smac protein levels in these tissues, with the aim to examine whether disturbed XIAP/Smac levels may be an indicator of neoadjuvant radio chemotherapy resistance. Expression of inhibitor of apoptosis proteins cIAP-1 and cIAP-2 was also examined. We found that levels of XIAP increased in accordance with the degree of radio chemotherapy resistance of the tissue. Levels of this protein were also significantly higher in tumour tissue, compared to matched normal tissue in highly resistant tissue. In contrast, Smac protein levels did not increase with radio chemotherapy resistance, and the protein was similarly expressed in normal and tumour tissue, indicating a shift in the balance of these proteins. Post treatment surgical resection tissue was available for 8 patients. When we compared matched tissue pre- and post- radio chemotherapy we found that XIAP levels increased significantly during treatment in both normal and tumour tissue, while Smac levels did not change. cIAP-1 and cIAP-2 levels were not differentially expressed in varying degrees of radio chemotherapy resistance, and neoadjuvant therapy did not alter expression of these proteins. These data indicate that disturbance of the XIAP/Smac balance may be a driver of radio chemotherapy resistance, and hence high levels of XIAP may

Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance.

Science.gov (United States)

Delgado, Asunción; Arco, Rocio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

2017-05-09

Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

Science.gov (United States)

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

Science.gov (United States)

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

Retinol binding protein 4, obesity, and insulin resistance in adolescents

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Ronaldi Noor

2017-02-01

Full Text Available Background Obesity is a global problem. Even in poor and developing countries, obesity has reached alarming levels. In childhood, obesity may lead to insulin resistance. Retinol binding protein (RBP4, secreted primarily by liver and adipose tissues, was recently proposed as a link between obesity and insulin resistance. The role of RBP4 in pediatric obesity and its relationship with insulin resistance have not been well elucidated. Objective To compare RBP4 levels in obese and lean adolescents and to assess for a relationship between RBP4 levels and insulin resistance. Method This cross-sectional study was conducted in three senior high schools in Padang, West Sumatera, Indonesia. Subjects were adolescents aged 14-18 years, who were obese or normal weight (n=56. We measured subjects’ body mass index (BMI and serum RBP4 concentrations. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR index. Results Similar RBP4 levels were found in the obese and normoweight groups (P>0.05. Higher RBP4 levels were found in the insulin resistant compared to the non-insulin resistant group, but the difference was not significant (P > 0.05. Conclusion There is no significant difference in mean RBP4 levels in obese adolescents compared to normoweight adolescents. Nor are mean RBP4 levels significantly different between obese adolescents with and without insulin resistance.

Identification of putative drug targets in Vancomycin-resistant Staphylococcus aureus (VRSA) using computer aided protein data analysis.

Science.gov (United States)

Hasan, Md Anayet; Khan, Md Arif; Sharmin, Tahmina; Hasan Mazumder, Md Habibul; Chowdhury, Afrin Sultana

2016-01-01

Vancomycin-resistant Staphylococcus aureus (VRSA) is a Gram-positive, facultative aerobic bacterium which is evolved from the extensive exposure of Vancomycin to Methicillin resistant S. aureus (MRSA) that had become the most common cause of hospital and community-acquired infections. Due to the emergence of different antibiotic resistance strains, there is an exigency to develop novel drug targets to address the provocation of multidrug-resistant bacteria. In this study, in-silico genome subtraction methodology was used to design potential and pathogen specific drug targets against VRSA. Our study divulged 1987 proteins from the proteome of 34,549 proteins, which have no homologues in human genome after sequential analysis through CD-HIT and BLASTp. The high stringency analysis of the remaining proteins against database of essential genes (DEG) resulted in 169 proteins which are essential for S. aureus. Metabolic pathway analysis of human host and pathogen by KAAS at the KEGG server sorted out 19 proteins involved in unique metabolic pathways. 26 human non-homologous membrane-bound essential proteins including 4 which were also involved in unique metabolic pathway were deduced through PSORTb, CELLO v.2.5, ngLOC. Functional classification of uncharacterized proteins through SVMprot derived 7 human non-homologous membrane-bound hypothetical essential proteins. Study of potential drug target against Drug Bank revealed pbpA-penicillin-binding protein 1 and hypothetical protein MQW_01796 as the best drug target candidate. 2D structure was predicted by PRED-TMBB, 3D structure and functional analysis was also performed. Protein-protein interaction network of potential drug target proteins was analyzed by using STRING. The identified drug targets are expected to have great potential for designing novel drugs against VRSA infections and further screening of the compounds against these new targets may result in the discovery of novel therapeutic compounds that can be

The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.

Science.gov (United States)

Fry, Bryan G; Roelants, Kim; Champagne, Donald E; Scheib, Holger; Tyndall, Joel D A; King, Glenn F; Nevalainen, Timo J; Norman, Janette A; Lewis, Richard J; Norton, Raymond S; Renjifo, Camila; de la Vega, Ricardo C Rodríguez

2009-01-01

Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase/prokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A(2), sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., vampire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition for venom. Given the large number of striking similarities between the protein compositions of conventional venoms and hematophagous secretions, we argue that the latter should also fall under the same definition.

Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

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Vesa Kirjavainen

Full Text Available Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS O-antigen (O-ag and outer core (OC do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp, an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

Penicillin-resistant viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains of Streptococcus pneumoniae.

OpenAIRE

Dowson, C G; Hutchison, A; Woodford, N; Johnson, A P; George, R C; Spratt, B G

1990-01-01

Penicillin-resistant strains of Streptococcus pneumoniae possess altered forms of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. The PBP2B genes of these strains have a mosaic structure, consisting of regions that are very similar to those in penicillin-sensitive strains, alternating with regions that are highly diverged. Penicillin-resistant strains of viridans groups streptococci (e.g., S. sanguis and S. oralis) that produce altered PBPs have also been reported. ...

Comparisons of protein profiles of beech bark disease resistant and susceptible American beech (Fagus grandifolia)

Science.gov (United States)

2013-01-01

Background Beech bark disease is an insect-fungus complex that damages and often kills American beech trees and has major ecological and economic impacts on forests of the northeastern United States and southeastern Canadian forests. The disease begins when exotic beech scale insects feed on the bark of trees, and is followed by infection of damaged bark tissues by one of the Neonectria species of fungi. Proteomic analysis was conducted of beech bark proteins from diseased trees and healthy trees in areas heavily infested with beech bark disease. All of the diseased trees had signs of Neonectria infection such as cankers or fruiting bodies. In previous tests reported elsewhere, all of the diseased trees were demonstrated to be susceptible to the scale insect and all of the healthy trees were demonstrated to be resistant to the scale insect. Sixteen trees were sampled from eight geographically isolated stands, the sample consisting of 10 healthy (scale-resistant) and 6 diseased/infested (scale-susceptible) trees. Results Proteins were extracted from each tree and analysed in triplicate by isoelectric focusing followed by denaturing gel electrophoresis. Gels were stained and protein spots identified and intensity quantified, then a statistical model was fit to identify significant differences between trees. A subset of BBD differential proteins were analysed by mass spectrometry and matched to known protein sequences for identification. Identified proteins had homology to stress, insect, and pathogen related proteins in other plant systems. Protein spots significantly different in diseased and healthy trees having no stand or disease-by-stand interaction effects were identified. Conclusions Further study of these proteins should help to understand processes critical to resistance to beech bark disease and to develop biomarkers for use in tree breeding programs and for the selection of resistant trees prior to or in early stages of BBD development in stands. Early

Effects of aging and insulin resistant states on protein anabolic responses in older adults.

Science.gov (United States)

Morais, Jose A; Jacob, Kathryn Wright; Chevalier, Stéphanie

2018-07-15

Insulin is the principal postprandial anabolic hormone and resistance to its action could contribute to sarcopenia. We developed different types of hyperinsulinemic clamp protocols to measure simultaneously glucose and protein metabolism in insulin resistant states (older adults, obesity, diabetes, etc.). To define effects of healthy aging in response to insulin, we employed the hyperinsulinemic, euglycemic and isoaminoacidemic (HYPER-1) clamp. The net whole-body anabolic (protein balance) response to hyperinsulinemia was lower in the elderly vs young (p = 0.007) and was highly correlated with the clamp glucose rate of disposal (r = 0.671, p anabolism compared with young ones. As most of the anabolism occurs during feeding, we studied the fed-state metabolic responses with aging using the hyperinsulinemic, hyperglycemic and hyperaminoacidemic clamp, including muscle biopsies. Older women showed comparable whole-body protein anabolic responses and stimulation of mixed-muscle protein synthesis by feeding to the young. The responses of skeletal muscle insulin signaling through the Akt-mTORC1 pathway were also unaltered, and therefore consistent with muscle protein synthesis results. Given that type 2 diabetes infers insulin resistance of protein metabolism with aging, we studied 10 healthy, 8 obese, and 8 obese type 2 diabetic elderly women using the HYPER-1 clamp. When compared to the group of young lean women to define the effects of obesity and diabetes with aging, whole-body change in net protein balance with hyperinsulinemia was similarly blunted in obese and diabetic older women. However, only elderly obese women with diabetes had lower net balance than lean older women. We conclude that with usual aging, the blunted whole-body anabolic response in elderly subjects is mediated by the failure of insulin to stimulate protein synthesis to the same extent as in the young, especially in men. This blunted response can be overcome at the whole-body and muscle

TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells

DEFF Research Database (Denmark)

Hekmat, Omid; Munk, Stephanie; Fogh, Louise

2013-01-01

may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated......Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass...... spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A and 2B, which...

Involvement of p38 mitogen-activated protein kinase in acquired gemcitabine-resistant human urothelial carcinoma sublines

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Yu-Ting Kao

2014-07-01

Full Text Available Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. A better understanding of how resistance arises and what molecular alterations correlate with resistance is the key to developing novel effective therapeutic strategies. To investigate the underlying mechanisms of gemcitabine (Gem resistance and provide possible therapeutic options, three Gem-resistant urothelial carcinoma sublines were established (NG0.6, NG0.8, and NG1.0. These cells were cross-resistant to arabinofuranosyl cytidine and cisplatin, but sensitive to 5-fluorouracil. The resistant cells expressed lower values of [hENT1 × dCK/RRM1 × RRM2] mRNA ratio. Two adenosine triphosphate-binding cassette proteins ABCD1 as well as multidrug resistance protein 1 were elevated. Moreover, cyclin D1, cyclin-dependent kinases 2 and 4 were upregulated, whereas extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK activity were repressed significantly. Administration of p38 MAPK inhibitor significantly reduced the Gem sensitivity in NTUB1 cells, whereas that of an extracellular signal-regulated kinase MAPK inhibitor did not. Furthermore, the Gem-resistant sublines also exhibited higher migration ability. Forced expression of p38 MAPK impaired the cell migration activity and augmented Gem sensitivity in NG1.0 cells. Taken together, these results demonstrate that complex mechanisms were merged in acquiring Gem resistance and provide information that can be important for developing therapeutic targets for treating Gem-resistant tumors.

The stress protein BAG3 stabilizes Mcl-1 protein and promotes survival of cancer cells and resistance to antagonist ABT-737.

Science.gov (United States)

Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D; Marnett, Lawrence J

2013-03-08

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.

The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737*

Science.gov (United States)

Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D.; Marnett, Lawrence J.

2013-01-01

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery. PMID:23341456

Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

KAUST Repository

Hussein, Rana

2012-11-01

Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization

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Tanne L. Cools

2017-11-01

Full Text Available HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea, is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA, which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP’s. Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A], we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP’s is reduced (≥74% and the antifungal activity of the variant is reduced (≥2-fold, highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1’s internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin.

Protein-protein interactions as a proxy to monitor conformational changes and activation states of the tomato resistance protein I-2

NARCIS (Netherlands)

Lukasik-Shreepaathy, E.; Vossen, J.H.; Tameling, W.I.L.; de Vroomen, M.J.; Cornelissen, B.J.C.; Takken, F.L.W.

2012-01-01

Plant resistance proteins (R) are involved in pathogen recognition and subsequent initiation of defence responses. Their activity is regulated by inter- and intramolecular interactions. In a yeast two-hybrid screen two clones (I2I-1 and I2I-2) specifically interacting with I-2, a Fusarium oxysporum

vPARP Adjusts MVP Expression in Drug-resistant Cell Lines in Conjunction with MDR Proteins.

Science.gov (United States)

Wojtowicz, Karolina; Januchowski, Radoslaw; Nowicki, Michal; Zabel, Maciej

2017-06-01

The definition of vault (ribonucleoprotein particles) function remains highly complex. Vaults may cooperate with multidrug resistance (MDR) proteins, supporting their role in drug resistance. This topic is the main theme of this publication. The cell viability was determined by an MTT assay. The protein expression was detected by western blot analysis. The proteins were knocked-down using siRNA. No major vault protein (MVP) in the LoVo/Dx and W1PR cell lines after tunicamycin treatment was shown. In W1PR cells with knocked-down MVP, a statistically significant decrease in cell viability was noted. In LoVo/Dx, W1TR and A2780TR cells were vault poly-ADP-ribose polymerase (vPARP) was knockdown, a decrease in cell viability was shown. Also, MVP silencing induced an increase in glycoprotein P (Pgp) expression in LoVo/Dx cells. MVP is important for the drug resistance of cancer cells, but it probably requires the presence of vPARP for full activation. Some correlations between MDR proteins and vaults exist. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Salivary gland proteome analysis reveals modulation of anopheline unique proteins in insensitive acetylcholinesterase resistant Anopheles gambiae mosquitoes.

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Sylvie Cornelie

Full Text Available Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1(R allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1(R resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.

Identification of a putative protein-profile associating with tamoxifen therapy-resistance in breast cancer

NARCIS (Netherlands)

A. Umar (Arzu); J.W.M. Martens (John); J.A. Foekens (John); L. Paša-Tolić (Ljiljana); H. Kang; A.M. Timmermans (Mieke); M.P. Look (Maxime); M.E. Meijer van Gelder (Marion); N. Jaitly (Navdeep); M.A. den Bakker (Michael)

2009-01-01

textabstractTamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical parameters can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards

Epithelial membrane protein-1 is a biomarker of gefitinib resistance.

Science.gov (United States)

Jain, Anjali; Tindell, Charles A; Laux, Isett; Hunter, Jacob B; Curran, John; Galkin, Anna; Afar, Daniel E; Aronson, Nina; Shak, Steven; Natale, Ronald B; Agus, David B

2005-08-16

We describe a molecular resistance biomarker to gefitinib, epithelial membrane protein-1 (EMP-1). Gefitinib is a small-molecule inhibitor that competes for the ATP-binding site on EGF receptor (EGFR) and has been approved for patients with advanced lung cancers. Treatment with gefitinib has resulted in clinical benefit in patients, and, recently, heterozygous somatic mutations within the EGFR catalytic domain have been identified as a clinical correlate to objective response to gefitinib. However, clinical resistance to gefitinib limits the utility of this therapeutic to a fraction of patients, and objective clinical responses are rare. We aimed to assess the molecular phenotype and mechanism of in vivo gefitinib resistance in xenograft models and in patient samples. We generated in vivo gefitinib-resistance models in an adenocarcinoma xenograft model by serially passaging tumors in nude mice in presence of gefitinib until resistance was acquired. EMP-1 was identified as a surface biomarker whose expression correlated with acquisition of gefitinib resistance. EMP-1 expression was further correlated with lack of complete or partial response to gefitinib in lung cancer patient samples as well as clinical progression to secondary gefitinib resistance. EMP-1 expression and acquisition of gefitinib clinical resistance was independent of gefitinib-sensitizing EGFR somatic mutations. This report suggests the role of the adhesion molecule, EMP-1, as a biomarker of gefitinib clinical resistance, and further suggests a probable cross-talk between this molecule and the EGFR signaling pathway.

Myofibrillar protein synthesis following ingestion of soy protein isolate at rest and after resistance exercise in elderly men

Directory of Open Access Journals (Sweden)

Yang Yifan

2012-06-01

Full Text Available Abstract Background Increased amino acid availability stimulates muscle protein synthesis, however, aged muscle appears less responsive to the anabolic effects of amino acids when compared to the young. We aimed to compare changes in myofibrillar protein synthesis (MPS in elderly men at rest and after resistance exercise following ingestion of different doses of soy protein and compare the responses to those we previously observed with ingestion of whey protein isolate. Methods Thirty elderly men (age 71 ± 5 y completed a bout of unilateral knee-extensor resistance exercise prior to ingesting no protein (0 g, or either 20 g or 40 g of soy protein isolate (0, S20, and S40 respectively. We compared these responses to previous responses from similar aged men who had ingested 20 g and 40 g of whey protein isolate (W20 and W40. A primed constant infusion of L-[1-13 C]leucine and L-[ring-13 C6]phenylalanine and skeletal muscle biopsies were used to measure whole-body leucine oxidation and MPS over 4 h post-protein consumption in both exercised and non-exercised legs. Results Whole-body leucine oxidation increased with protein ingestion and was significantly greater for S20 vs. W20 (P = 0.003. Rates of MPS for S20 were less than W20 (P = 0.02 and not different from 0 g (P = 0.41 in both exercised and non-exercised leg muscles. For S40, MPS was also reduced compared with W40 under both rested and post-exercise conditions (both P P = 0.04. Conclusions The relationship between protein intake and MPS is both dose and protein source-dependent, with isolated soy showing a reduced ability, as compared to isolated whey protein, to stimulate MPS under both rested and post-exercise conditions. These differences may relate to the lower postprandial leucinemia and greater rates of amino acid oxidation following ingestion of soy versus whey protein.

A wheat cinnamyl alcohol dehydrogenase TaCAD12 contributes to host resistance to the sharp eyespot disease

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Wei Rong

2016-11-01

Full Text Available Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, is a destructive disease in hexaploid wheat (Triticum aestivum L.. In Arabidopsis, certain cinnamyl alcohol dehydrogenases (CADs have been implicated in monolignol biosynthesis and in defense response to bacterial pathogen infection. However, little is known about CADs in wheat defense responses to necrotrophic or soil-borne pathogens. In this study, we isolate a wheat CAD gene TaCAD12 in response to R. cerealis infection through microarray-based comparative transcriptomics, and study the enzyme activity and defense role of TaCAD12 in wheat. The transcriptional levels of TaCAD12 in sharp eyespot-resistant wheat lines were significantly higher compared with those in susceptible wheat lines. The sequence and phylogenetic analyses revealed that TaCAD12 belongs to IV group in CAD family. The biochemical assay proved that TaCAD12 protein is an authentic CAD enzyme and possesses catalytic efficiencies towards both coniferyl aldehyde and sinapyl aldehyde. Knock-down of TaCAD12 transcript significantly repressed resistance of the gene-silenced wheat plants to sharp eyespot caused by R. cerealis, whereas TaCAD12 overexpression markedly enhanced resistance of the transgenic wheat lines to sharp eyespot. Furthermore, certain defense genes (Defensin, PR10, PR17c, and Chitinase1 and monolignol biosynthesis-related genes (TaCAD1, TaCCR, and TaCOMT1 were up-regulated in the TaCAD12-overexpressing wheat plants but down-regulated in TaCAD12-silencing plants. These results suggest that TaCAD12 positively contributes to resistance against sharp eyespot through regulation of the expression of certain defense genes and monolignol biosynthesis-related genes in wheat.

C-reactive protein levels and treatment resistance in schizophrenia - A Danish population-based cohort study

DEFF Research Database (Denmark)

Horsdal, Henriette Thisted; Wimberley, Theresa; Benros, Michael Eriksen

2017-01-01

-time schizophrenia diagnosis and a baseline C-reactive protein measurement (a commonly available marker of systemic inflammation) from 2000 to 2012. We defined treatment resistance as the earliest observed instance of either clozapine initiation or hospital admission due to schizophrenia after having received......OBJECTIVE: Schizophrenia is associated with increased levels of inflammatory markers. However, it remains unclear whether inflammatory markers are associated with treatment-resistant schizophrenia. METHODS: We conducted a population-based follow-up study among individuals with a first...... (4.0 vs. 3.1 mg/L, p = .13) was observed among the 52 (13.3%) treatment-resistant individuals. Increased levels of C-reactive protein (above 3 mg/L) at baseline were not associated with treatment resistance (adjusted hazard ratio = 0.99, 95% confidence interval [0.56, 1.73]). CONCLUSIONS: C...

Application of protein typing in molecular epidemiological investigation of nosocomial infection outbreak of aminoglycoside-resistant Pseudomonas aeruginosa.

Science.gov (United States)

Song, Min; Tang, Min; Ding, Yinghuan; Wu, Zecai; Xiang, Chengyu; Yang, Kui; Zhang, Zhang; Li, Baolin; Deng, Zhenghua; Liu, Jinbo

2017-12-16

Pseudomonas aeruginosan has emerged as an important pathogen elated to serious infections and nosocomial outbreaks worldwide. This study was conducted to understand the prevalence of aminoglycoside (AMG)-resistant P. aeruginosa in our hospital and to provide a scientific basis for control measures against nosocomial infections. Eighty-two strains of P. aeruginosa were isolated from clinical departments and divided into AMG-resistant strains and AMG-sensitive strains based on susceptibility test results. AMG-resistant strains were typed by drug resistance gene typing (DRGT) and protein typing. Five kinds of aminoglycoside-modifying enzyme (AME) genes were detected in the AMG-resistant group. AMG-resistant P. aeruginosa strains were classified into three types and six subtypes by DRGT. Four protein peaks, namely, 9900.02, 7600.04, 9101.25 and 10,372.87 Da, were significantly and differentially expressed between the two groups. AMG-resistant P. aeruginosa strains were also categorised into three types and six subtypes at the distance level of 10 by protein typing. AMG-resistant P. aeruginosa was cloned spread in our hospital; the timely implementation of nosocomial infection prevention and control strategies were needed in preventing outbreaks and epidemic of AMG-resistant P. aeruginosa. SELDI-TOF MS technology can be used for bacterial typing, which provides a new method of clinical epidemiological survey and nosocomial infection control.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

Directory of Open Access Journals (Sweden)

Prajna Jena

Full Text Available Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

Analysis of the protein profiles of the antibiotic-resistant Salmonella ...

African Journals Online (AJOL)

The emergent Salmonella typhimurium definitive phage type (DT) 104 is of particular global concern due to its frequent isolation and multiple antibiotic resistances. There is thus a need to know the kind of proteins expressed by S. typhimurium DT104 so as to provide a basis for developing an intervention. This study ...

Host-plant-mediated effects of Nadefensin on herbivore and pathogen resistance in Nicotiana attenuata

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Baldwin Ian T

2008-10-01

Full Text Available Abstract Background The adage from Shakespeare, "troubles, not as single spies, but in battalions come," holds true for Nicotiana attenuata, which is commonly attacked by both pathogens (Pseudomonas spp. and herbivores (Manduca sexta in its native habitats. Defense responses targeted against the pathogens can directly or indirectly influence the responses against the herbivores. Nadefensin is an effective induced defense gene against the bacterial pathogen Pseudomonas syringae pv tomato (PST DC3000, which is also elicited by attack from M. sexta larvae, but whether this defense protein influences M. sexta's growth and whether M. sexta-induced Nadefensin directly or indirectly influences PST DC3000 resistance are unknown. Results M. sexta larvae consumed less on WT and on Nadefensin-silenced N. attenuata plants that had previously been infected with PST DC3000 than on uninfected plants. WT plants infected with PST DC3000 showed enhanced resistance to PST DC3000 and decreased leaf consumption by M. sexta larvae, but larval mass gain was unaffected. PST DC3000-infected Nadefensin-silenced plants were less resistant to subsequent PST DC3000 challenge, and on these plants, M. sexta larvae consumed less and gained less mass. WT and Nadefensin-silenced plants previously damaged by M. sexta larvae were better able to resist subsequent PST DC3000 challenges than were undamaged plants. Conclusion These results demonstrate that Na-defensin directly mediates defense against PST DC3000 and indirectly against M. sexta in N. attenuata. In plants that were previously infected with PST DC3000, the altered leaf chemistry in PST DC3000-resistant WT plants and PST DC3000-susceptible Nadefensin-silenced plants differentially reduced M. sexta's leaf consumption and mass gain. In plants that were previously damaged by M. sexta, the combined effect of the altered host plant chemistry and a broad spectrum of anti-herbivore induced metabolomic responses was more

Human Skeletal Muscle Stem Cells in Adaptations to Exercise; Effects of Resistance Exercise Contraction Mode and Protein Supplementation

DEFF Research Database (Denmark)

Farup, Jean

2014-01-01

the effect of contraction mode specific resistance training and protein supplementation on whole muscle and tendon hypertrophy. Quadriceps muscle and patellar tendon cross-sectional area (CSA) was quantified using magnetic resonance imaging pre and post 12 weeks of eccentric (Ecc) or concentric (Conc...... concentric resistance training and ingestion of protein influence myocellular adaptations, with special emphasis on muscle stem cell adaptations, during both acute and prolonged resistance exercise in human skeletal muscle. Paper I. Whey protein supplementation accelerates satellite cell proliferation during...... recovery from eccentric exercise In paper I, we evaluated the effect of a single bout of unaccustomed eccentric exercise on fiber type specific SC content by immunohistochemistry. Subjects received either hydrolysed whey protein (Whey) or iso-caloric carbohydrate (Placebo) in the days post eccentric...

Physiological quality and gene expression related to heat-resistant proteins at different stages of development of maize seeds.

Science.gov (United States)

Andrade, T; Von Pinho, E V R; Von Pinho, R G; Oliveira, G E; Andrade, V; Fernandes, J S

2013-09-13

We quantified and characterized the expression of heat-resistant proteins during seed development of maize lines with distinct levels of tolerance to high drying temperature. A corn field was planted for multiplication of seeds of different lines, two tolerant and two non-tolerant to high drying temperatures. Harvest of the seeds was carried out at various stages of development and they were then subjected to tests of moisture content, germination, first count of germination, accelerated aging, and cold test. The seeds were stored in a freezer for later analysis of expression of heat-resistant proteins by means of real-time PCR, electrophoresis, and spectrophotometry. We observed that heat-resistant proteins are expressed in a differential manner in seeds from different lines and at different stages of development. The expression of heat-resistant proteins was earlier in lines tolerant to high drying temperatures. Greater germination and vigor values was found for seeds collected at the last stage of development.

Synovial Fluid α-Defensin as a Biomarker for Peri-Prosthetic Joint Infection: A Systematic Review and Meta-Analysis.

Science.gov (United States)

Li, Bin; Chen, Fei; Liu, Yi; Xu, Guokang

Total joint arthroplasty (TJA) has been one of the most beneficial interventions for treating patients suffering from joint disorders. However, peri-prosthetic joint infection (PJI) is a serious complication that often accompanies TJA and the diagnosis of PJI is remains difficult. Questions remain regarding whether certain biomarkers can be valuable in the diagnosis of PJI. We conducted our systematic review by searching PubMed, Embase, Web of Science, the Cochrane Library, and Science Direct with the key words "periprosthetic joint infection," "synovial fluid," and "α-defensin." Studies that provided sufficient data to construct 2 × 2 contingency tables were chosen based on inclusion and exclusion criteria. The quality of included studies was assessed according to the revised Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) criteria. The pooled sensitivity, specificity, and diagnostic odds ratio (DOR) were calculated for the included studies. The summary receiver operating characteristic (SROC) curve and the area under the summary receiver operating characteristic (AUSROC) were used to evaluate the overall diagnostic performance. Eight studies were included in this systematic review. Among them four articles were included in meta-analysis. A total of 421 participants were studied in the meta-analysis. The pooled sensitivity, specificity, and DOR were 0.98 (95% confidence interval [CI]: 0.94-1.00), 0.97 (95% CI: 0.95-0.99), and 1095.49 (95% CI: 283.68.58-4230.45), respectively. The AUSROC was 0.9949 (standard error [SE] 0.0095). Synovial fluid α-defensin is a biomarker of high sensitivity and specificity for the diagnosis of PJI.

Identifying the Proteins that Mediate the Ionizing Radiation Resistance of Deinococcus Radiodurans R1

Energy Technology Data Exchange (ETDEWEB)

Battista, John R

2010-02-22

The primary objectives of this proposal was to define the subset of proteins required for the ionizing radiation (IR) resistance of Deinococcus radiodurans R1, characterize the activities of those proteins, and apply what was learned to problems of interest to the Department of Energy.

Configurable Resistive Switching between Memory and Threshold Characteristics for Protein-Based Devices

KAUST Repository

Wang, Hong; Du, Yuanmin; Li, Yingtao; Zhu, Bowen; Leow, Wan Ru; Li, Yuangang; Pan, Jisheng; Wu, Tao; Chen, Xiaodong

2015-01-01

The employ of natural biomaterials as the basic building blocks of electronic devices is of growing interest for biocompatible and green electronics. Here, resistive switching (RS) devices based on naturally silk protein with configurable

Expression of multidrug resistance-related protein (MRP-1), lung resistance-related protein (LRP) and topoisomerase-II (TOPO-II) in Wilms' tumor: immunohistochemical study using TMA methodology.

Science.gov (United States)

Fridman, Eduard; Skarda, Jozef; Pinthus, Jonatan H; Ramon, Jonathan; Mor, Yoran

2008-06-01

MRP-1, LRP and TOPO-II are all associated with protection of the cells from the adverse effects of various chemotherapeutics. The aim of this study was to measure the expression of these proteins in Wilms' tumor (WT). TMA block was constructed from 14 samples of WT's and from xenografts derived from them. Sections of the TMA were used for immunostaining against MRP-1, LRP and TOPO-IIa. All normal kidneys expressed MRP-1 but were either weakly or negatively stained for LRP and TOPO-IIa. In WT samples, MRP-1 was universally expressed, exclusively in the tubular component, while there was no expression of LRP and TOPO-IIa showed heterogeneous distribution. The xenografts varied in their MRP-1 and TOPO-IIa expression and exhibited weak/negative staining of LRP. This study shows that although all the proteins evaluated, had different expression patterns in the tumor samples, the most prominent changes in expression were found for MRP-1. The exact clinical implications of these changes in expression and their relevance to the resistance of these tumors to chemotherapy requires further investigation. The finding of different expression profiles for the multidrug resistance proteins in the original WT's and their xenografts suggests that the results of animal cancer models may be difficult to interpret.

Configurable Resistive Switching between Memory and Threshold Characteristics for Protein-Based Devices

KAUST Repository

Wang, Hong

2015-05-01

The employ of natural biomaterials as the basic building blocks of electronic devices is of growing interest for biocompatible and green electronics. Here, resistive switching (RS) devices based on naturally silk protein with configurable functionality are demonstrated. The RS type of the devices can be effectively and exactly controlled by controlling the compliance current in the set process. Memory RS can be triggered by a higher compliance current, while threshold RS can be triggered by a lower compliance current. Furthermore, two types of memory devices, working in random access and WORM modes, can be achieved with the RS effect. The results suggest that silk protein possesses the potential for sustainable electronics and data storage. In addition, this finding would provide important guidelines for the performance optimization of biomaterials based memory devices and the study of the underlying mechanism behind the RS effect arising from biomaterials. Resistive switching (RS) devices with configurable functionality based on protein are successfully achieved. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanosensitive molecular networks involved in transducing resistance exercise-signals into muscle protein accretion

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Emil Rindom

2016-11-01

Full Text Available Loss of skeletal muscle myofibrillar protein with disease and/or inactivity can severely deteriorate muscle strength and function. Strategies to counteract wasting of muscle myofibrillar protein are therefore desirable and invite for considerations on the potential superiority of specific modes of resistance exercise and/or the adequacy of low load resistance exercise regimens as well as underlying mechanisms. In this regard, delineation of the potentially mechanosensitive molecular mechanisms underlying muscle protein synthesis (MPS, may contribute to understanding on how differentiated resistance exercise can transduce a mechanical signal into stimulation of muscle accretion. Recent findings suggest specific upstream exercise-induced mechano-sensitive myocellular signaling pathways to converge on mammalian target of rapamycin complex 1 (mTORC1, to influence MPS. This may e.g. implicate mechanical activation of signaling through a diacylglycerol kinase (DGKζ-phosphatidic acid (PA axis or implicate integrin deformation to signal through a Focal adhesion kinase (FAK-Tuberous Sclerosis Complex 2TSC2-Ras homolog enriched in brain (Rheb axis. Moreover, since initiation of translation is reliant on mRNA, it is also relevant to consider potentially mechanosensitive signaling pathways involved in muscle myofibrillar gene transcription and whether some of these pathways converge with those affecting mTORC1 activation for MPS. In this regard, recent findings suggest how mechanical stress may implicate integrin deformation and/or actin dynamics to signal through a Ras homolog gene family member A protein (RhoA-striated muscle activator of Rho signaling (STARS axis or how it may implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP signaling through a small mother of decapentaplegic (Smad axis.

Comparative genomics of Fusarium oxysporum f. sp. melonis reveals the secreted protein recognized by the Fom-2 resistance gene in melon

NARCIS (Netherlands)

Schmidt, S.M.; Lukasiewicz, J.; Farrer, R.; van Dam, P.; Bertoldo, C.; Rep, M.

Development of resistant crops is the most effective way to control plant diseases to safeguard food and feed production. Disease resistance is commonly based on resistance genes, which generally mediate the recognition of small proteins secreted by invading pathogens. These proteins secreted by

Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans

DEFF Research Database (Denmark)

Esmarck, B.; Andersen, J.L.; Olsen, S.

2001-01-01

1. Age-associated loss of skeletal muscle mass and strength can partly be counteracted by resistance training, causing a net synthesis of muscular proteins. Protein synthesis is influenced synergistically by postexercise amino acid supplementation, but the importance of the timing of protein intake...

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

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Luftig Ronald

2007-09-01

Full Text Available Abstract Background Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines. Results HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b. Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein. Conclusion This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.

Production of low-affinity penicillin-binding protein by low- and high-resistance groups of methicillin-resistant Staphylococcus aureus.

Science.gov (United States)

Murakami, K; Nomura, K; Doi, M; Yoshida, T

1987-01-01

Methicillin- and cephem-resistant Staphylococcus aureus (137 strains) for which the cefazolin MICs are at least 25 micrograms/ml could be classified into low-resistance (83% of strains) and high-resistance (the remaining 17%) groups by the MIC of flomoxef (6315-S), a 1-oxacephalosporin. The MICs were less than 6.3 micrograms/ml and more than 12.5 micrograms/ml in the low- and high-resistance groups, respectively. All strains produced penicillin-binding protein 2' (PBP 2'), which has been associated with methicillin resistance and which has very low affinity for beta-lactam antibiotics. Production of PBP 2' was regulated differently in low- and high-resistance strains. With penicillinase-producing strains of the low-resistance group, cefazolin, cefamandole, and cefmetazole induced PBP 2' production about 5-fold, while flomoxef induced production 2.4-fold or less. In contrast, penicillinase-negative variants of low-resistance strains produced PBP 2' constitutively in large amounts and induction did not occur. With high-resistance strains, flomoxef induced PBP 2' to an extent similar to that of cefazolin in both penicillinase-producing and -negative strains, except for one strain in which the induction did not occur. The amount of PBP 2' induced by beta-lactam antibiotics in penicillinase-producing strains of the low-resistance group correlated well with resistance to each antibiotic. Large amounts of PBP 2' in penicillinase-negative variants of the low-resistance group did not raise the MICs of beta-lactam compounds, although these strains were more resistant when challenged with flomoxef for 2 h. Different regulation of PBP 2' production was demonstrated in the high- and low-resistance groups, and factor(s) other than PBP 2' were suggested to be involved in the methicillin resistance of high-resistance strains. Images PMID:3499861

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

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Anna C Llewellyn

Full Text Available Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI, validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and

Effects of Insect Protein Supplementation during Resistance Training on Changes in Muscle Mass and Strength in Young Men

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Mathias T. Vangsoe

2018-03-01

Full Text Available During prolonged resistance training, protein supplementation is known to promote morphological changes; however, no previous training studies have tested the effect of insect protein isolate in a human trial. The aim of this study was to investigate the potential effect of insect protein as a dietary supplement to increase muscle hypertrophy and strength gains during prolonged resistance training in young men. Eighteen healthy young men performed resistance training four day/week for eight weeks. Subjects were block randomized into two groups consuming either an insect protein isolate or isocaloric carbohydrate supplementation within 1 h after training and pre-sleep on training days. Strength and body composition were measured before and after intervention to detect adaptions to the resistance training. Three-day weighed dietary records were completed before and during intervention. Fat- and bone- free mass (FBFM improved significantly in both groups (Mean (95% confidence interval (CI, control group (Con: (2.5 kg (1.5, 3.5 p < 0.01, protein group (Pro: (2.7 kg (1.6, 3.8 p < 0.01 from pre- to post-. Leg and bench press one repetition maximum (1 RM improved by Con: (42.0 kg (32.0, 52.0 p < 0.01 and (13.8 kg (10.3, 17.2 p < 0.01, Pro: (36.6 kg (27.3, 45.8 p < 0.01 and (8.1 kg (4.5, 11.8 p < 0.01, respectively. No significant differences in body composition and muscle strength improvements were found between groups. In young healthy men, insect protein supplementation did not improve adaptations to eight weeks of resistance training in comparison to carbohydrate supplementation. A high habitual protein intake in both Con and Pro may partly explain our observation of no superior effect of insect protein supplementation.

Damage-recognition proteins as a potential indicator of DNA-damage-mediated sensitivity or resistance of human cells to ultraviolet radiation

International Nuclear Information System (INIS)

Chao, C.C.-K.

1992-01-01

The authors compared damage-recognition proteins in cells expressing different sensitivities to DNA damage. An increase in damage-recognition proteins and an enhancement of plasmid re-activation were detected in HeLa cells resistant to cisplatin and u.v. However, repair-defective cells derived from xeroderma-pigmentosum (a rare skin disease) patients did not express less cisplatin damage-recognition proteins than repair-competent cells, suggesting that damage-recognition-protein expression may not be related to DNA repair. By contrast, cells resistant to DNA damage consistently expressed high levels of u.v.-modified-DNA damage-recognition proteins. The results support the notion that u.v. damage-recognition proteins are different from those that bind to cisplatin. Findings also suggest that the damage-recognition proteins identified could be used as potential indicators of the sensitivity or resistance of cells to u.v. (author)

Improving protein resistance of {alpha}-Al{sub 2}O{sub 3} membranes by modification with POEGMA brushes

Energy Technology Data Exchange (ETDEWEB)

He Huating [State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009 (China); Jing Wenheng, E-mail: jingwenheng@yahoo.com.cn [State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009 (China); Xing Weihong; Fan Yiqun [State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009 (China)

2011-11-15

A kind of protein-resistant ceramic membrane is prepared by grafting poly(oligo (ethylene glycol) methyl ether methacrylate) (POEGMA) brushes onto the surfaces and pore walls of {alpha}-Al{sub 2}O{sub 3} membrane (AM) by surface-initiated atom-transfer radical polymerization (SI-ATRP). Contact-angle, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and field-emission scanning electron microscopy (FESEM) were measured to confirm that the surfaces and pore walls of the ceramic porous membranes have been modified by the brushes with this method successfully. The protein interaction behavior with the POEGMA modified membranes (AM-POEGMA) was studied by the model protein of bovine serum albumin (BSA). A protein-resistant mechanism of AM-POEGMA was proposed to describe an interesting phenomenon discovered in the filtration experiment, in which the initial flux filtrating BSA solution is higher than the pure water flux. The fouling of AM-POEGMA was easier to remove than AM for the action of POEGMA brushes, indicated that the ceramic porous membranes modified with POEGMA brushes exhibit excellent protein resistance.

Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise.

Science.gov (United States)

Hector, Amy J; McGlory, Chris; Damas, Felipe; Mazara, Nicole; Baker, Steven K; Phillips, Stuart M

2018-01-01

Preservation of lean body mass (LBM) may be important during dietary energy restriction (ER) and requires equal rates of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Currently, the relative contribution of MPS and MPB to the loss of LBM during ER in humans is unknown. We aimed to determine the impact of dietary protein intake and resistance exercise on MPS and MPB during a controlled short-term energy deficit. Adult men (body mass index, 28.6 ± 0.6 kg/m 2 ; age 22 ± 1 yr) underwent 10 d of 40%-reduced energy intake while performing unilateral resistance exercise and consuming lower protein (1.2 g/kg/d, n = 12) or higher protein (2.4 g/kg/d, n = 12). Pre- and postintervention testing included dual-energy X-ray absorptiometry, primed constant infusion of ring -[ 13 C 6 ]phenylalanine, and 15 [N]phenylalanine to measure acute postabsorptive MPS and MPB; D 2 O to measure integrated MPS; and gene and protein expression. There was a decrease in acute MPS after ER (higher protein, 0.059 ± 0.006 to 0.051 ± 0.009%/h; lower protein, 0.061 ± 0.005 to 0.045 ± 0.006%/h; P resistance exercise (higher protein, 0.067 ± 0.01%/h; lower protein, 0.061 ± 0.006%/h), and integrated MPS followed a similar pattern. There was no change in MPB (energy balance, 0.080 ± 0.01%/hr; ER rested legs, 0.078 ± 0.008%/hr; ER exercised legs, 0.079 ± 0.006%/hr). We conclude that a reduction in MPS is the main mechanism that underpins LBM loss early in ER in adult men.-Hector, A. J., McGlory, C., Damas, F., Mazara, N., Baker, S. K., Phillips, S. M. Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise. © FASEB.

Regulation of Multidrug Resistance Proteins by Genistein in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity

OpenAIRE

Rigalli, Juan Pablo; Ciriaci, Nadia; Arias, Agostina; Ceballos, Mar?a Paula; Villanueva, Silvina Stella Maris; Luquita, Marcelo Gabriel; Mottino, Aldo Domingo; Ghanem, Carolina In?s; Catania, Viviana Alicia; Ruiz, Mar?a Laura

2015-01-01

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of t...

The effects of whey protein with or without carbohydrates on resistance training adaptations.

Science.gov (United States)

Hulmi, Juha J; Laakso, Mia; Mero, Antti A; Häkkinen, Keijo; Ahtiainen, Juha P; Peltonen, Heikki

2015-01-01

Nutrition intake in the context of a resistance training (RT) bout may affect body composition and muscle strength. However, the individual and combined effects of whey protein and carbohydrates on long-term resistance training adaptations are poorly understood. A four-week preparatory RT period was conducted in previously untrained males to standardize the training background of the subjects. Thereafter, the subjects were randomized into three groups: 30 g of whey proteins (n = 22), isocaloric carbohydrates (maltodextrin, n = 21), or protein + carbohydrates (n = 25). Within these groups, the subjects were further randomized into two whole-body 12-week RT regimens aiming either for muscle hypertrophy and maximal strength or muscle strength, hypertrophy and power. The post-exercise drink was always ingested immediately after the exercise bout, 2-3 times per week depending on the training period. Body composition (by DXA), quadriceps femoris muscle cross-sectional area (by panoramic ultrasound), maximal strength (by dynamic and isometric leg press) and serum lipids as basic markers of cardiovascular health, were analysed before and after the intervention. Twelve-week RT led to increased fat-free mass, muscle size and strength independent of post-exercise nutrient intake (P carbohydrate group independent of the type of RT (P carbohydrate group (P carbohydrates or combination of proteins and carbohydrates did not have a major effect on muscle size or strength when ingested two to three times a week. However, whey proteins may increase abdominal fat loss and relative fat-free mass adaptations in response to resistance training when compared to fast-acting carbohydrates.

The effects of whey protein with or without carbohydrates on resistance training adaptations

OpenAIRE

Hulmi, Juha; Laakso, Mia; Mero, Antti; Häkkinen, Keijo; Ahtiainen, Juha; Peltonen, Heikki

2015-01-01

Background: Nutrition intake in the context of a resistance training (RT) bout may affect body composition and muscle strength. However, the individual and combined effects of whey protein and carbohydrates on long-term resistance training adaptations are poorly understood. Methods: A four-week preparatory RT period was conducted in previously untrained males to standardize the training background of the subjects. Thereafter, the subjects were randomized into three groups: 30 g of...

Comprehensive assessment of sequence variation within the copy number variable defensin cluster on 8p23 by target enriched in-depth 454 sequencing

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Zhang Xinmin

2011-05-01

Full Text Available Abstract Background In highly copy number variable (CNV regions such as the human defensin gene locus, comprehensive assessment of sequence variations is challenging. PCR approaches are practically restricted to tiny fractions, and next-generation sequencing (NGS approaches of whole individual genomes e.g. by the 1000 Genomes Project is confined by an affordable sequence depth. Combining target enrichment with NGS may represent a feasible approach. Results As a proof of principle, we enriched a ~850 kb section comprising the CNV defensin gene cluster DEFB, the invariable DEFA part and 11 control regions from two genomes by sequence capture and sequenced it by 454 technology. 6,651 differences to the human reference genome were found. Comparison to HapMap genotypes revealed sensitivities and specificities in the range of 94% to 99% for the identification of variations. Using error probabilities for rigorous filtering revealed 2,886 unique single nucleotide variations (SNVs including 358 putative novel ones. DEFB CN determinations by haplotype ratios were in agreement with alternative methods. Conclusion Although currently labor extensive and having high costs, target enriched NGS provides a powerful tool for the comprehensive assessment of SNVs in highly polymorphic CNV regions of individual genomes. Furthermore, it reveals considerable amounts of putative novel variations and simultaneously allows CN estimation.

Effects of whey protein supplement in the elderly submitted to resistance training: systematic review and meta-analysis.

Science.gov (United States)

Colonetti, Tamy; Grande, Antonio Jose; Milton, Karen; Foster, Charlie; Alexandre, Maria Cecilia Manenti; Uggioni, Maria Laura Rodrigues; Rosa, Maria Inês da

2017-05-01

We performed a systematic review to map the evidence and analyze the effect of whey protein supplementation in the elderly submitted to resistance training. A comprehensive search on Medline, LILACS, EMBASE, and the Cochrane Library for relevant publications was conducted until August 2015. The terms used in the search were: "Resistance training"; "Whey protein"; "Elderly". A total of 632 studies were screened. Five studies were included composing a sample of 391 patients. The supplement whey protein was associated with higher total protein ingestion 9.40 (95% CI: 4.03-14.78), and with an average change in plasma leucine concentration. The supplementation was also associated with increased mixed muscle protein synthesis 1.26 (95% CI: 0.46-2.07) compared to the control group. We observed an increase in total protein intake, resulting in increased concentration of leucine and mixed muscle protein fractional synthesis rate.

Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

Science.gov (United States)

Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

2011-11-01

Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

KAUST Repository

Phelan, Jody

2016-03-23

Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance

Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress.

Science.gov (United States)

Karlsson, Markus; Kurz, Tino

2016-09-01

Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress. The sensitivity of ARPE-19 cells to H2 O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4 , heat shock or FeCl3 , as well as siRNA-mediated downregulation of the same proteins. Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2 O2 . This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins. The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

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Rong Wang

Full Text Available The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco, horseradish peroxidase (HRP, hemoglobin (Hb], and two pepsin resistant proteins [lipid transfer protein (LTP and soybean trypsin inhibitor (STI]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to

Recruitment of a penicillin-binding protein gene from Neisseria flavescens during the emergence of penicillin resistance in Neisseria meningitidis

OpenAIRE

SPRATT, BG; ZHANG, QY; JONES, DM; HUTCHISON, A; BRANNIGAN, JA; DOWSON, CG

1989-01-01

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria meningitidis produce altered forms of penicillin-binding protein 2 that have decreased affinity for penicillin. The sequence of the penicillin-binding protein 2 gene (penA) from a penicillin-resistant strain of N. meningitidis was compared to the sequence of the same gene from penicillin-sensitive strains and from penicillin-sensitive and penicillin-resistant strains of Neisseria gonorrhoeae. The penA genes from penicilli...

Protein networks in induced sputum from smokers and COPD patients

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Baraniuk JN

2015-09-01

Ig joining chain compared to other groups. Emphysema subjects’ sputum was unique, with high plasma proteins and components of neutrophil extracellular traps, such as histones and defensins. In contrast, defensins were correlated with epithelial proteins in all other groups. Protein-network interactions were unique to each group. Conclusion: The proteomes were interpreted as complex “biosignatures” that suggest distinct pathophysiological mechanisms for mucin 5AC hypersecretion, airflow obstruction, and inflammatory emphysema phenotypes. Proteomic phenotyping may improve genotyping studies by selecting more homogeneous study groups. Each phenotype may require its own mechanistically based diagnostic, risk-assessment, drug- and other treatment algorithms. Keywords: cigarette smokers, chronic bronchitis, emphysema, proteomics, mucous hypersecretion, mucin 5AC, neutrophil extracellular nets

Pokeweed Antiviral Protein: Its Cytotoxicity Mechanism and Applications in Plant Disease Resistance

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Rong Di

2015-03-01

Full Text Available Pokeweed antiviral protein (PAP is a 29 kDa type I ribosome inactivating protein (RIP found in pokeweed plants. Pokeweed produces different forms of PAP. This review focuses on the spring form of PAP isolated from Phytolacca americana leaves. PAP exerts its cytotoxicity by removing a specific adenine from the α-sarcin/ricin loop of the large ribosomal RNA. Besides depurination of the rRNA, PAP has additional activities that contribute to its cytotoxicity. The mechanism of PAP cytotoxicity is summarized based on evidence from the analysis of transgenic plants and the yeast model system. PAP was initially found to be anti-viral when it was co-inoculated with plant viruses onto plants. Transgenic plants expressing PAP and non-toxic PAP mutants have displayed broad-spectrum resistance to both viral and fungal infection. The mechanism of PAP-induced disease resistance in transgenic plants is summarized.

Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

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Charlene Desbonnet

2016-04-01

Full Text Available The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins” is reliant on the presence of class A penicillin-binding proteins (Pbps PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2 of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP. Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.

Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892

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Rudolf, Jeffrey D. [Scripps Research Inst., Jupiter, FL (United States); Bigelow, Lance [Argonne National Lab. (ANL), Argonne, IL (United States); Chang, Changsoo [Argonne National Lab. (ANL), Argonne, IL (United States); Cuff, Marianne E. [Argonne National Lab. (ANL), Argonne, IL (United States); Lohman, Jeremy R. [Scripps Research Inst., Jupiter, FL (United States); Chang, Chin-Yuan [Scripps Research Inst., Jupiter, FL (United States); Ma, Ming [Scripps Research Inst., Jupiter, FL (United States); Yang, Dong [Scripps Research Inst., Jupiter, FL (United States); Clancy, Shonda [Argonne National Lab. (ANL), Argonne, IL (United States); Babnigg, Gyorgy [Argonne National Lab. (ANL), Argonne, IL (United States); Joachimiak, Andrzej [Argonne National Lab. (ANL), Argonne, IL (United States); Phillips, George N. [Rice Univ., Houston, TX (United States); Shen, Ben [Scripps Research Inst., Jupiter, FL (United States)

2015-11-17

The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 angstrom, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.

Defensin-like ZmES4 mediates pollen tube burst in maize via opening of the potassium channel KZM1.

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Suseno Amien

2010-06-01

Full Text Available In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K(+-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K(+ channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K(+ influx. We further suggest that K(+ influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization

Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1

Science.gov (United States)

Márton, Mihaela L.; Debener, Thomas; Geiger, Dietmar; Becker, Dirk; Dresselhaus, Thomas

2010-01-01

In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K+-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K+ channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K+ influx. We further suggest that K+ influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a

Decreased UV light resistance of spores of Bacillus subtilis strains deficient in pyrimidine dimer repair and small, acid-soluble spore proteins

International Nuclear Information System (INIS)

Setlow, B.; Setlow, P.

1988-01-01

Loss of small, acid-soluble spore protein alpha reduced spore UV resistance 30- to 50-fold in Bacillus subtilis strains deficient in pyrimidine dimer repair, but gave only a 5- to 8-fold reduction in UV resistance in repair-proficient strains. However, both repair-proficient and -deficient spores lacking this protein had identical heat and gamma-radiation resistance

Bioinformatics and structural characterization of a hypothetical protein from Streptococcus mutans: implication of antibiotic resistance.

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Jie Nan

2009-10-01

Full Text Available As an oral bacterial pathogen, Streptococcus mutans has been known as the aetiologic agent of human dental caries. Among a total of 1960 identified proteins within the genome of this organism, there are about 500 without any known functions. One of these proteins, SMU.440, has very few homologs in the current protein databases and it does not fall into any protein functional families. Phylogenetic studies showed that SMU.440 is related to a particular ecological niche and conserved specifically in some oral pathogens, due to lateral gene transfer. The co-occurrence of a MarR protein within the same operon among these oral pathogens suggests that SMU.440 may be associated with antibiotic resistance. The structure determination of SMU.440 revealed that it shares the same fold and a similar pocket as polyketide cyclases, which indicated that it is very likely to bind some polyketide-like molecules. From the interlinking structural and bioinformatics studies, we have concluded that SMU.440 could be involved in polyketide-like antibiotic resistance, providing a better understanding of this hypothetical protein. Besides, the combination of multiple methods in this study can be used as a general approach for functional studies of a protein with unknown function.

Ectopic expression of X-linked lymphocyte-regulated protein pM1 renders tumor cells resistant to antitumor immunity.

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Kang, Tae Heung; Noh, Kyung Hee; Kim, Jin Hee; Bae, Hyun Cheol; Lin, Ken Y; Monie, Archana; Pai, Sara I; Hung, Chien-Fu; Wu, T-C; Kim, Tae Woo

2010-04-15

Tumor immune escape is a major obstacle in cancer immunotherapy, but the mechanisms involved remain poorly understood. We have previously developed an immune evasion tumor model using an in vivo immune selection strategy and revealed Akt-mediated immune resistance to antitumor immunity induced by various cancer immunotherapeutic agents. In the current study, we used microarray gene analysis to identify an Akt-activating candidate molecule overexpressed in immune-resistant tumors compared with parental tumors. X-linked lymphocyte-regulated protein pM1 (XLR) gene was the most upregulated in immune-resistant tumors compared with parental tumor cells. Furthermore, the retroviral transduction of XLR in parental tumor cells led to activation of Akt, resulting in upregulation of antiapoptotic proteins and the induction of immune resistance phenotype in parental tumor cells. In addition, we found that transduction of parental tumor cells with other homologous genes from the mouse XLR family, such as synaptonemal complex protein 3 (SCP3) and XLR-related, meiosis-regulated protein (XMR) and its human counterpart of SCP3 (hSCP3), also led to activation of Akt, resulting in the upregulation of antiapoptotic proteins and induction of immune resistance phenotype. Importantly, characterization of a panel of human cervical cancers revealed relatively higher expression levels of hSCP3 in human cervical cancer tissue compared with normal cervical tissue. Thus, our data indicate that ectopic expression of XLR and its homologues in tumor cells represents a potentially important mechanism for tumor immune evasion and serves as a promising molecular target for cancer immunotherapy. (c) 2010 AACR.

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins.

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Helliwell, Emily E; Vega-Arreguín, Julio; Shi, Zi; Bailey, Bryan; Xiao, Shunyuan; Maximova, Siela N; Tyler, Brett M; Guiltinan, Mark J

2016-03-01

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Towards predictive resistance models for agrochemicals by combining chemical and protein similarity via proteochemometric modelling.

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van Westen, Gerard J P; Bender, Andreas; Overington, John P

2014-10-01

Resistance to pesticides is an increasing problem in agriculture. Despite practices such as phased use and cycling of 'orthogonally resistant' agents, resistance remains a major risk to national and global food security. To combat this problem, there is a need for both new approaches for pesticide design, as well as for novel chemical entities themselves. As summarized in this opinion article, a technique termed 'proteochemometric modelling' (PCM), from the field of chemoinformatics, could aid in the quantification and prediction of resistance that acts via point mutations in the target proteins of an agent. The technique combines information from both the chemical and biological domain to generate bioactivity models across large numbers of ligands as well as protein targets. PCM has previously been validated in prospective, experimental work in the medicinal chemistry area, and it draws on the growing amount of bioactivity information available in the public domain. Here, two potential applications of proteochemometric modelling to agrochemical data are described, based on previously published examples from the medicinal chemistry literature.

Partly replacing meat protein with soy protein alters insulin resistance and blood lipids in postmenopausal women with abdominal obesity

NARCIS (Netherlands)

Nielen, van M.; Feskens, E.J.M.; Rietman, A.; Siebelink, E.; Mensink, M.R.

2014-01-01

Increasing protein intake and soy consumption appear to be promising approaches to prevent metabolic syndrome (MetS). However, the effect of soy consumption on insulin resistance, glucose homeostasis, and other characteristics of MetS is not frequently studied in humans. We aimed to investigate the

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

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Catarina Moreirinha

2018-03-01

Full Text Available The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells.

Binding site alteration is responsible for field-isolated resistance to Bacillus thuringiensis Cry2A insecticidal proteins in two Helicoverpa species.

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Silvia Caccia

Full Text Available BACKGROUND: Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F(2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac. METHODOLOGY/PRINCIPAL FINDINGS: Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with (125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in (125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins. CONCLUSION/SIGNIFICANCE: This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported

Anti-Restriction Protein, KlcAHS, Promotes Dissemination of Carbapenem Resistance

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Xiaofei Jiang

2017-05-01

Full Text Available Carbapenemase-producing Klebsiella pneumoniae (KPC has emerged and spread throughout the world. A retrospective analysis was performed on carbapenem-resistant K. pneumoniae isolated at our teaching hospital during the period 2009–2010, when the initial outbreak occurred. To determine the mechanism(s that underlies the increased infectivity exhibited by KPC, Multilocus Sequence Typing (MLST was conducted. A series of plasmids was also extracted, sequenced and analyzed. Concurrently, the complete sequences of blaKPC−2-harboring plasmids deposited in GenBank were summarized and aligned. The blaKPC−2 and KlcAHS genes in the carbapenem-resistant K. pneumoniae isolates were examined. E. coli strains, carrying different Type I Restriction and Modification (RM systems, were selected to study the interaction between RM systems, anti-RM systems and horizontal gene transfer (HGT. The ST11 clone predominated among 102 carbapenem-resistant K. pneumoniae isolates, all harbored the blaKPC−2 gene; 98% contained the KlcAHS gene. KlcAHS was one of the core genes in the backbone region of most blaKPC−2 carrying plasmids. Type I RM systems in the host bacteria reduced the rate of pHS10842 plasmid transformation by 30- to 40-fold. Presence of the anti-restriction protein, KlcAHS, on the other hand, increased transformation efficiency by 3- to 6-fold. These results indicate that RM systems can significantly restrict HGT. In contrast, KlcAHS can disrupt the RM systems and promote HGT by transformation. These findings suggest that the anti-restriction protein, KlcAHS, represents a novel mechanism that facilitates the increased transfer of blaKPC-2 and KlcAHS-carrying plasmids among K. pneumoniae strains.

P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

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Loll-Krippleber, Raphael; Brown, Grant W

2017-09-15

mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

Metabolic responses to high protein diet in Korean elite bodybuilders with high-intensity resistance exercise

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Choue Ryowon

2011-07-01

Full Text Available Abstract Background High protein diet has been known to cause metabolic acidosis, which is manifested by increased urinary excretion of nitrogen and calcium. Bodybuilders habitually consumed excessive dietary protein over the amounts recommended for them to promote muscle mass accretion. This study investigated the metabolic response to high protein consumption in the elite bodybuilders. Methods Eight elite Korean bodybuilders within the age from 18 to 25, mean age 21.5 ± 2.6. For data collection, anthropometry, blood and urinary analysis, and dietary assessment were conducted. Results They consumed large amounts of protein (4.3 ± 1.2 g/kg BW/day and calories (5,621.7 ± 1,354.7 kcal/day, as well as more than the recommended amounts of vitamins and minerals, including potassium and calcium. Serum creatinine (1.3 ± 0.1 mg/dl and potassium (5.9 ± 0.8 mmol/L, and urinary urea nitrogen (24.7 ± 9.5 mg/dl and creatinine (2.3 ± 0.7 mg/dl were observed to be higher than the normal reference ranges. Urinary calcium (0.3 ± 0.1 mg/dl, and phosphorus (1.3 ± 0.4 mg/dl were on the border of upper limit of the reference range and the urine pH was in normal range. Conclusions Increased urinary excretion of urea nitrogen and creatinine might be due to the high rates of protein metabolism that follow high protein intake and muscle turnover. The obvious evidence of metabolic acidosis in response to high protein diet in the subjects with high potassium intake and intensive resistance exercise were not shown in this study results. However, this study implied that resistance exercise with adequate mineral supplementation, such as potassium and calcium, could reduce or offset the negative effects of protein-generated metabolic changes. This study provides preliminary information of metabolic response to high protein intake in bodybuilders who engaged in high-intensity resistance exercise. Further studies will be needed to determine the effects of the intensity

Lipopolysaccharide-binding protein: localization in secretory granules of Paneth cells in the mouse small intestine

DEFF Research Database (Denmark)

Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte

2009-01-01

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host's response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study...... in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58-60 kDa protein of LBP distinctly labeled a small population of cells located deep...... into the crypts. This cell population was also positive for lysozyme and alpha-defensin 4, identifying Paneth cells as the main intestinal LBP-producing cells. By immunogold electron microscopy, intense labeling was observed in the secretory granules of these cells. We conclude that Paneth cells express LBP...

Association of ERCC1 protein expression to platinum resistance in epithelial ovarian cancer

DEFF Research Database (Denmark)

Dahl Steffensen, Karina; Waldstrøm, Marianne; Jakobsen, Anders

was to investigate if immunohistochemical expression of ERCC1 protein was associated with resistance to standard combination carboplatin and paclitaxel chemotherapy in newly diagnosed ovarian cancer patients. Methods: Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian...

Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents.

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Ryan J Mailloux

2010-10-01

Full Text Available Uncoupling protein-2 (UCP2 is known to suppress mitochondrial reactive oxygen species (ROS production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

Autophagy is induced by resistance exercise in young men but unfolded protein response is induced regardless of age.

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Hentilä, Jaakko; Ahtiainen, Juha P; Paulsen, Gøran; Raastad, Truls; Häkkinen, Keijo; Mero, Antti A; Hulmi, Juha J

2018-04-02

Autophagy and unfolded protein response (UPR) appear to be important for skeletal muscle homeostasis and may be altered by exercise. Our aim was to investigate the effects of resistance exercise and training on indicators of UPR and autophagy in healthy untrained young men (n = 12, 27 ± 4 years) and older men (n = 8, 61 ± 6 years) as well as in resistance-trained individuals (n = 15, 25 ± 5 years). Indicators of autophagy and UPR were investigated from the muscle biopsies after a single resistance exercise bout and after 21 weeks of resistance training. Lipidated LC3II as an indicator of autophagosome content increased at 48 hours post resistance exercise (P resistance-training period (P resistance exercise in untrained young and older men (P resistance-training period regardless of age. UPR was unchanged within the first few hours after the resistance exercise bout regardless of the training status. Changes in autophagy and UPR ER indicators did not correlate with a resistance-training-induced increase in muscle strength and size. Autophagosome content is increased by resistance training in young previously untrained men, but this response may be blunted by aging. However, unfolded protein response is induced by an unaccustomed resistance exercise bout in a delayed manner regardless of age. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Initial infection of roots and leaves reveals different resistance phenotypes associated with coat protein gene-mediated resistance to Potato mop-top virus.

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Germundsson, Anna; Sandgren, Maria; Barker, Hugh; Savenkov, Eugene I; Valkonen, Jari P T

2002-05-01

Resistance to the pomovirus Potato mop-top virus (PMTV) was studied in potato (Solanum tuberosum cv. Saturna) and Nicotiana benthamiana transformed with the coat protein (CP) gene of PMTV. The incidence of PMTV infections was reduced in tubers of the CP-transgenic potatoes grown in the field in soil infested with the viruliferous vector, Spongospora subterranea. However, in those tubers that were infected, all three virus RNAs were detected and virus titres were high. The CP-transgenic N. benthamiana plants were inoculated with PMTV using two methods. Following mechanical inoculation of leaves, no RNA 3 (the CP-encoding RNA homologous to the transgene) was detected in leaves, but in some plants low amounts of RNA 3 were detected in roots; RNA 2 was readily detected in leaves and roots of several plants. Inoculation of roots using viruliferous S. subterranea resulted in infection of roots in all plants and the three PMTV RNAs were detected. However, no systemic movement of PMTV from roots to the above-ground parts was observed, indicating a novel expression of resistance. These data indicate that the CP gene-mediated resistance to PMTV specifically restricts accumulation of PMTV RNA 3, and is more effective in leaves than roots. Furthermore, expression of resistance is different depending on whether leaves or roots are inoculated. Data do not exclude the possibility that both a protein-mediated and an RNA-mediated resistance mechanism are involved.

A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

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Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

2012-01-01

Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

Science.gov (United States)

Fenyk, Stepan; Campillo, Alba de San Eustaquio; Pohl, Ehmke; Hussey, Patrick J; Cann, Martin J

2012-02-03

Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

Pea proteins oral supplementation promotes muscle thickness gains during resistance training: a double-blind, randomized, Placebo-controlled clinical trial vs. Whey protein.

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Babault, Nicolas; Païzis, Christos; Deley, Gaëlle; Guérin-Deremaux, Laetitia; Saniez, Marie-Hélène; Lefranc-Millot, Catherine; Allaert, François A

2015-01-01

The effects of protein supplementation on muscle thickness and strength seem largely dependent on its composition. The current study aimed at comparing the impact of an oral supplementation with vegetable Pea protein (NUTRALYS®) vs. Whey protein and Placebo on biceps brachii muscle thickness and strength after a 12-week resistance training program. One hundred and sixty one males, aged 18 to 35 years were enrolled in the study and underwent 12 weeks of resistance training on upper limb muscles. According to randomization, they were included in the Pea protein (n = 53), Whey protein (n = 54) or Placebo (n = 54) group. All had to take 25 g of the proteins or placebo twice a day during the 12-week training period. Tests were performed on biceps muscles at inclusion (D0), mid (D42) and post training (D84). Muscle thickness was evaluated using ultrasonography, and strength was measured on an isokinetic dynamometer. Results showed a significant time effect for biceps brachii muscle thickness (P Pea, Whey and Placebo, respectively; P Pea group as compared to Placebo whereas there was no difference between Whey and the two other conditions. Muscle strength also increased with time with no statistical difference between groups. In addition to an appropriate training, the supplementation with pea protein promoted a greater increase of muscle thickness as compared to Placebo and especially for people starting or returning to a muscular strengthening. Since no difference was obtained between the two protein groups, vegetable pea proteins could be used as an alternative to Whey-based dietary products. The present trial has been registered at ClinicalTrials.gov (NCT02128516).

Coexistence of protease sensitive and resistant prion protein in 129VV homozygous sporadic Creutzfeldt–Jakob disease: a case report

Directory of Open Access Journals (Sweden)

Rodríguez-Martínez Ana B

2012-10-01

Full Text Available Abstract Introduction The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported. Case presentation A 74-year-old Caucasian woman showed a sporadic Creutzfeldt–Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77. Neuropathological findings showed: spongiform change in the patient’s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous. Conclusion Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt–Jakob disease. This highlights the

Determination of proteins induced in response to jasmonic acid and salicylic acid in resistant and susceptible cultivars of tomato.

Science.gov (United States)

Afroz, Amber; Khan, Muhammad Rashid; Komatsu, Setsuko

2010-07-01

Jasmonic acid (JA) and salicylic acid (SA) are signaling molecules that play key roles in the regulation of metabolic processes, reproduction, and defense against pathogens. The proteomics approach was used to identify proteins that are induced by JA and SA in the tomato cultivars Roma and Pant Bahr, which are susceptible and resistant to bacterial wilt, respectively. Threonine deaminase and leucine amino peptidase were upregulated, and ribulose-1,5-bisphosphate carboxylase/oxygenase small chain was downregulated by time-course application of JA. Translationally controlled tumor protein was upregulated by time-course application of SA. Protein disulfide isomerase was upregulated by application of either JA or SA. Proteins related to defense, energy, and protein destination/storage are suspected to be responsible for the susceptibility or resistance of the cultivars. Furthermore, in Roma, iron ABC transporter was upregulated by JA and down-regulated by SA. Iron ABC transporter plays a part in the signal transduction of both JA and SA in cultivars of tomato that are resistant to bacterial wilt.

Natural coagulation inhibitors and active protein c resistance in preeclampsia

Directory of Open Access Journals (Sweden)

Cengiz Demir

2010-01-01

Full Text Available INTRODUCTION: The etiology of preeclampsia is not fully established. A few studies have shown a relationship between natural coagulation inhibitors and preeclampsia. OBJECTIVES: The purpose of this study was to investigate the status of natural coagulation inhibitors and active protein C resistance (APC-R in preeclampsia. PATIENTS AND METHODS: We studied 70 women with preeclampsia recruited consecutively and 70 healthy pregnant and 70 nonpregnant women as controls. Plasma protein C (PC, free protein S (fPS, antithrombin III (ATIII and APC-R were evaluated. RESULTS: ATIII values were found to be significantly lower in preeclamptic patients than in the control groups (p< 0.001. Nevertheless, there was no significant difference between the healthy pregnant and nonpregnant women groups (p=0.141. The fPS values of the preeclamptic and healthy pregnant groups were lower than that of the nonpregnant group (p< 0.001, and the fPS value of the preeclamptic pregnant women was lower than that of healthy pregnant women (p<0.001. The PC value of the preeclamptic pregnant women was lower than that of the control groups (p< 0.001. The PC value of the healthy pregnant women was lower than that of the nonpregnant women (p< 0.001. The mean APC activity values were lower in the preeclamptic patients than that of the control groups (p< 0.001, p< 0.001. The APC-R positivity rates of the preeclamptic groups were higher than that of the control groups (p<0.001. CONCLUSIONS: This study demonstrated that ATIII, fPS, PC values and APC resistance were lower and APC-R positivity was higher in preeclamptic women than in normal pregnant and nonpregnant women.

Detection of the Mr 110,000 lung resistance-related protein LRP/MVP with monoclonal antibodies.

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Schroeijers, A B; Scheffer, G L; Reurs, A W; Pijnenborg, A C; Abbondanza, C; Wiemer, E A; Scheper, R J

2001-11-01

The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR.

Diagnosis of prosthetic joint infection with alpha-defensin using a lateral flow device: a multicentre study.

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Berger, P; Van Cauter, M; Driesen, R; Neyt, J; Cornu, O; Bellemans, J

2017-09-01

The purpose of this current multicentre study is to analyse the presence of alpha-defensin proteins in synovial fluid using the Synovasure lateral flow device and to determine its diagnostic reliability and accuracy compared with the prosthetic joint infection (PJI) criteria produced by the Musculoskeletal Infection Society (MSIS). A cohort of 121 patients comprising 85 total knee arthroplasties and 36 total hip arthroplasties was prospectively evaluated between May 2015 and June 2016 in three different orthopaedic centres. The tests were performed on patients with a chronically painful prosthesis undergoing a joint aspiration in a diagnostic pathway or during revision surgery. Based on the MSIS criteria, 34 patients (28%) would have had a PJI, and 87 patients had no PJI. Testing with the lateral flow device had a sensitivity of 97.1% (95% confidence intervals (CI) 84.5 to 99.9) and a specificity of 96.6% (95% CI 90.3 to 99.2). The positive predictive value was 91.7% (95% CI 77.7% to 98.3), and the negative predictive value was 98.8% (95% CI 93.6 to 99.9). Receiver operator characteristics analysis demonstrated an area under the curve for the Synovasure test of 0.97 (95% CI 0.93 to 1.00). Our findings suggest that the Synovasure test has an excellent diagnostic performance to confirm or reject the diagnosis of a PJI. The results are promising for the care of the painful or problematic knee and hip joint arthroplasty and the test should be considered as part of the diagnostic toolbox for PJIs. Cite this article: Bone Joint J 2017;99-B:1176-82. ©2017 The British Editorial Society of Bone & Joint Surgery.

[The role of Cd-binding proteins and phytochelatins in the formation of cadmium resistance in Nicotiana plumbaginifolia cell lines].

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Fenik, S I; Solodushko, V G; Kaliniak, T B; Blium, Ia B

2007-01-01

Nicotiana plumbaginifolia callus lines with the equal resistance to cadmium have been produced under different selective conditions--either without inhibition of the phytochelatin synthesis (line Cd-R) or in the presence of the inhibitor butionine sulfoximine (line Cd-Ri). The level of phytochelatin synthesis in the line Cd-R five-fold exceeded the control value and in the line Cd-Ri it was twice as much as in the control. It was shown that in the control line mainly three cadmium-binding proteins are expressed of the molecular weihgts 41, 34 and 19 kD. The common feature of the both resistant lines is the expression of the cadmium-binding proteins of 40, 37 and 19 kD. The resistant lines differ with respect to the synthesis of relatively low-molecular cadmium-binding proteins. The proteins of the molecular weights 12.5, 11.5 and 9 kD are expressed in the line Cd-R, while the proteins of 13 and 10 kD are expressed in the line Cd-Ri. It was supposed that both the phytochelatins and the Cd-binding proteins contribute to the resisitance of N. plumbaginifolia callus lines to cadmium and the lack of the phytochelatins can be equilibrated by the changes in the low-molecular Cd-binding protein synthesis.

Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions

NARCIS (Netherlands)

Breedveld, Pauline; Zelcer, Noam; Pluim, Dick; Sönmezer, Ozgür; Tibben, Matthijs M.; Beijnen, Jos H.; Schinkel, Alfred H.; van Tellingen, Olaf; Borst, Piet; Schellens, Jan H. M.

2004-01-01

The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an

Protein cross-linking, peroxidase and beta-1,3-endoglucanase involved in resistance of pea against Orobanche crenata.

Science.gov (United States)

Pérez-de-Luque, Alejandro; González-Verdejo, Clara I; Lozano, M Dolores; Dita, Miguel A; Cubero, José I; González-Melendi, Pablo; Risueño, María C; Rubiales, Diego

2006-01-01

Root holoparasitic angiosperms, like Orobanche spp, completely lack chlorophyll and totally depend on their host for their supply of nutrients. O. crenata is a severe constraint to the cultivation of legumes and breeding for resistance remains the most economical, feasible, and environmentally friendly method of control. Due to the lack of resistance in commercial pea cultivars, the use of wild relatives for breeding is necessary, and an understanding of the mechanisms underlying host resistance is needed in order to improve screening for resistance in breeding programmes. Compatible and incompatible interactions between O. crenata and pea have been studied using cytochemical procedures. The parasite was stopped in the host cortex before reaching the central cylinder, and accumulation of H2O2, peroxidases, and callose were detected in neighbouring cells. Protein cross-linking in the host cell walls appears as the mechanism of defence, halting penetration of the parasite. In situ hybridization studies have also shown that a peroxidase and a beta-glucanase are differently expressed in cells of the resistant host (Pf651) near the penetration point. The role of these proteins in the resistance to O. crenata is discussed.

High-resolution structure of the antibiotic resistance protein NimA from Deinococcus radiodurans

International Nuclear Information System (INIS)

Leiros, Hanna-Kirsti S.; Tedesco, Consiglia; McSweeney, Seán M.

2008-01-01

In this paper, the 1.2 Å atomic resolution crystal structure of the 5-nitroimidazole antibiotic resistance protein NimA from Deinococcus radiodurans (DrNimA) is presented. Many anaerobic human pathogenic bacteria are treated using 5-nitroimidazole-based (5-Ni) antibiotics, a class of inactive prodrugs that contain a nitro group. The nitro group must be activated in an anaerobic one-electron reduction and is therefore dependent on the redox system in the target cells. Antibiotic resistance towards 5-Ni drugs is found to be related to the nim genes (nimA, nimB, nimC, nimD, nimE and nimF), which are proposed to encode a reductase that is responsible for converting the nitro group of the antibiotic into a nonbactericidal amine. A mechanism for the Nim enzyme has been proposed in which two-electron reduction of the nitro group leads to the generation of nontoxic derivatives and confers resistance against these antibiotics. The cofactor was found to be important in the mechanism and was found to be covalently linked to the reactive His71. In this paper, the 1.2 Å atomic resolution crystal structure of the 5-nitroimidazole antibiotic resistance protein NimA from Deinococcus radiodurans (DrNimA) is presented. A planar cofactor is clearly visible and well defined in the electron-density map adjacent to His71, the identification of the cofactor and its properties are discussed

Molecular basis of glyphosate resistance: Different approaches through protein engineering

Science.gov (United States)

Pollegioni, Loredano; Schonbrunn, Ernst; Siehl, Daniel

2011-01-01

Glyphosate (N-phosphonomethyl-glycine) is the most-used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple small molecule is mainly due to the high specificity of glyphosate towards the plant enzyme enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway leading to biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced thus allowing the application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on the evolution of mechanisms of resistance to glyphosate as obtained through natural diversity, the gene shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer rationale for the means by which the modifications made have had their intended effect. PMID:21668647

Ghrelin- and GH-induced insulin resistance: no association with retinol-binding protein-4

DEFF Research Database (Denmark)

Vestergaard, Esben Thyssen; Krag, Morten B; Poulsen, Morten M

2013-01-01

Supraphysiological levels of ghrelin and GH induce insulin resistance. Serum levels of retinol-binding protein-4 (RBP4) correlate inversely with insulin sensitivity in patients with type 2 diabetes. We aimed to determine whether ghrelin and GH affect RBP4 levels in human subjects....

Detection of First-Line Drug Resistance Mutations and Drug-Protein Interaction Dynamics from Tuberculosis Patients in South India.

Science.gov (United States)

Nachappa, Somanna Ajjamada; Neelambike, Sumana M; Amruthavalli, Chokkanna; Ramachandra, Nallur B

2018-05-01

Diagnosis of drug-resistant tuberculosis predominantly relies on culture-based drug susceptibility testing, which take weeks to produce a result and a more time-efficient alternative method is multiplex allele-specific PCR (MAS-PCR). Also, understanding the role of mutations in causing resistance helps better drug designing. To evaluate the ability of MAS-PCR in the detection of drug resistance and to understand the mechanism of interaction of drugs with mutant proteins in Mycobacterium tuberculosis. Detection of drug-resistant mutations using MAS-PCR and validation through DNA sequencing. MAS-PCR targeted five loci on three genes, katG 315 and inhA -15 for the drug isoniazid (INH), and rpoB 516, 526, and 531 for rifampicin (RIF). Furthermore, the sequence data were analyzed to study the effect on interaction of the anti-TB drug molecule with the target protein using in silico docking. We identified drug-resistant mutations in 8 out of 114 isolates with 2 of them as multidrug-resistant TB using MAS-PCR. DNA sequencing confirmed only six of these, recording a sensitivity of 85.7% and specificity of 99.3% for MAS-PCR. Molecular docking showed estimated free energy of binding (ΔG) being higher for RIF binding with RpoB S531L mutant. Codon 315 in KatG does not directly interact with INH but blocks the drug access to active site. We propose DNA sequencing-based drug resistance detection for TB, which is more accurate than MAS-PCR. Understanding the action of resistant mutations in disrupting the normal drug-protein interaction aids in designing effective drug alternatives.

Comparison of protein profiles of beech bark disease-resistant or beech bark disease-susceptible American beech

Science.gov (United States)

Mary E. Mason; Marek Krasowski; Judy Loo; Jennifer. Koch

2011-01-01

Proteomic analysis of beech bark proteins from trees resistant and susceptible to beech bark disease (BBD) was conducted. Sixteen trees from eight geographically isolated stands, 10 resistant (healthy) and 6 susceptible (diseased/infested) trees, were studied. The genetic complexity of the sample unit, the sampling across a wide geographic area, and the complexity of...

Anti-protein C antibodies are associated with resistance to endogenous protein C activation and a severe thrombotic phenotype in antiphospholipid syndrome.

Science.gov (United States)

Arachchillage, D R J; Efthymiou, M; Mackie, I J; Lawrie, A S; Machin, S J; Cohen, H

2014-11-01

Antiphospholipid antibodies may interfere with the anticoagulant activity of activated protein C (APC) to induce acquired APC resistance (APCr). To investigate the frequency and characteristics of APCr by using recombinant human APC (rhAPC) and endogenous protein C activation in antiphospholipid syndrome (APS). APCr was assessed in APS and non-APS venous thromboembolism (VTE) patients on warfarin and normal controls with rhAPC or Protac by thrombin generation. IgG anti-protein C and anti-protein S antibodies and avidity were assessed by ELISA. APS patients showed greater resistance to both rhAPC and Protac than non-APS patients and normal controls (median normalized endogenous thrombin potential inhibition): APS patients with rhAPC, 81.3% (95% confidence interval [CI] 75.2-88.3%; non-APS patients with rhAPC, 97.7% (95% CI 93.6-101.8%; APS patients with Protac, 66.0% (95% CI 59.5-72.6%); and non-APS patients with Protac, 80.7 (95% CI 74.2-87.2%). APS patients also had a higher frequency and higher levels of anti-protein C antibodies, with 60% (15/25) high-avidity antibodies. High-avidity anti-protein C antibodies were associated with greater APCr and with a severe thrombotic phenotype (defined as the development of recurrent VTE while patients were receiving therapeutic anticoagulation or both venous and arterial thrombosis). Twelve of 15 (80%) patients with high-avidity anti-protein C antibodies were classified as APS category I. Thrombotic APS patients showed greater APCr to both rhAPC and activation of endogenous protein C by Protac. High-avidity anti-protein C antibodies, associated with greater APCr, may provide a marker for a severe thrombotic phenotype in APS. However, in patients with category I APS, it remains to be established whether anti-protein C or anti-β2 -glycoprotein I antibodies are responsible for APCr. © 2014 International Society on Thrombosis and Haemostasis.

Dual regulatory roles of the extended N terminus for activation of the tomato MI-1.2 resistance protein

NARCIS (Netherlands)

Lukasik-Shreepaathy, E.; Slootweg, E.; Richter, H.; Goverse, A.; Cornelissen, B.J.C.; Takken, F.L.W.

2012-01-01

Plant resistance (R) proteins mediate race-specific immunity and initiate host defenses that are often accompanied by a localized cell-death response. Most R proteins belong to the nucleotide binding-leucine-rich repeat (NB-LRR) protein family, as they carry a central NB-ARC domain fused to an LRR

Proteomic analysis of the maize rachis: potential roles of constitutive and induced proteins in resistance to Aspergillus flavus infection and aflatoxin accumulation.

Science.gov (United States)

Pechanova, Olga; Pechan, Tibor; Williams, W Paul; Luthe, Dawn S

2011-01-01

Infection of the maize (Zea mays L.) with aflatoxigenic fungus Aspergillus flavus and consequent contamination with carcinogenic aflatoxin is a persistent and serious agricultural problem causing disease and significant crop losses worldwide. The rachis (cob) is an important structure of maize ear that delivers essential nutrients to the developing kernels and A. flavus spreads through the rachis to infect kernels within the ear. Therefore, rachis plays an important role in fungal proliferation and subsequent kernel contamination. We used proteomic approaches and investigated the rachis tissue from aflatoxin accumulation resistant (Mp313E and Mp420) and susceptible (B73 and SC212m) maize inbred lines. First, we compared rachis proteins from resistant and susceptible inbred lines, which revealed that the young resistant rachis contains higher levels of abiotic stress-related proteins and proteins from phenylpropanoid metabolism, whereas susceptible young rachis contains pathogenesis-related proteins, which are generally inducible upon biotic stress. Second, we identified A. flavus-responsive proteins in rachis of both resistant and susceptible genotypes after 10- and 35-day infection. Differential expression of many stress/defense proteins during rachis juvenility, maturation and after A. flavus challenge demonstrates that resistant rachis relies on constitutive defenses, while susceptible rachis is more dependent on inducible defenses. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

NARCIS (Netherlands)

Bossers, A.; Belt, P.B.G.M.; Raymond, G.J.; Caughey, B.; Vries, de R.; Smits, M.

1997-01-01

Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrPSc) of the host-encoded cellular prion protein (PrPC) mainly in the central nervous system.

Expression of avian β-defensins and Toll-like receptor genes in the rooster epididymis during growth and Salmonella infection.

Science.gov (United States)

Anastasiadou, M; Avdi, M; Michailidis, G

2013-08-01

The epididymis is an organ involved in the maturation, transport, and storage of sperm prior to ejaculation. As epididymis is exposed to a constant risk of inflammatory conditions that may lead to transient or permanent sterility, protection of this organ from pathogens is an essential aspect of reproductive physiology. The families of antimicrobial peptides β-defensins and the pattern-recognition receptors Toll-like (TLR) mediate innate immunity in various vertebrates including avian species. As rooster infertility is a major concern in the poultry industry, the objectives of this study were to determine the expression profile of the entire family of the avian β-defensins (AvBD) and TLR genes in the rooster epididymis, to investigate whether sexual maturation affects their epididymidal mRNA abundance and to determine the changes in their expression levels in response to Salmonella enteritidis (SE) infection in the epididymis of sexually mature roosters. RNA was extracted from the epididymis of healthy pubertal, sexually mature and aged birds, and from sexually mature SE infected birds. RT-PCR analysis revealed that 10 members of the AvBD and nine members of the TLR gene families were expressed in the epididymis. Quantitative real-time PCR analysis revealed that the epididymidal mRNA abundance of certain AvBD and TLR genes was developmentally regulated with respect to sexual maturation. SE infection resulted in a significant induction of AvBD 1, 9, 10, 12 and 14, as well as TLR 1-2, 2-1, 2-2, 4, 5 and 7 genes, in the epididymis of sexually mature roosters, compared to healthy birds of the same age. These findings provide strong evidence to suggest that the rooster epididymis is capable of initiating an inflammatory response to Salmonella, through activation of certain members of the AvBD and TLR gene families. Copyright © 2013 Elsevier B.V. All rights reserved.

Overexpression of protein kinase A - RIalpha reduces lipofection efficiency of cisplatin-resistant human tumor cells.

Science.gov (United States)

Son, K K; Rosenblatt, J

2001-04-10

Cisplatin-resistant variant A2780CP/vector cells were 4.0-5.3-fold more transfectable and 7.6-fold more resistant to cisplatin than their parent cisplatin-sensitive human ovarian carcinoma A2780/vector cells. Overexpression of cAMP-dependent protein kinase Type I regulatory alpha subunit (PKA-RIalpha) gene in A2780CP cells significantly reduced (maximum 47.0%) the transfection activity, with a slight reduction (maximum 27.3%) of cisplatin resistance, of A2780CP cells. However, RIalpha-overexpressing A2780CP (A2780CP/RIalpha) cells were still 2.5-to 3.0-fold more transfectable and 5.5-fold more resistant to cisplatin than A2780 cells. This results suggest that gene transfer efficiency is associated with cisplatin resistance, in part, through the PKA-mediated cAMP signal transduction pathway.

Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil.

Science.gov (United States)

Bernardi, Daniel; Salmeron, Eloisa; Horikoshi, Renato Jun; Bernardi, Oderlei; Dourado, Patrick Marques; Carvalho, Renato Assis; Martinelli, Samuel; Head, Graham P; Omoto, Celso

2015-01-01

Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F2 screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105

Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

Science.gov (United States)

Camera, Donny M; Burniston, Jatin G; Pogson, Mark A; Smiles, William J; Hawley, John A

2017-12-01

It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise. Precursor enrichment and the rate of incorporation of deuterium oxide into newly synthesized muscle proteins were determined by mass spectrometry. Ninety proteins were included in the DPP, with 28 proteins exhibiting significant responses to REX. The most common pattern of response was an increase in turnover, followed by an increase in abundance with no detectable increase in protein synthesis. Here, we provide novel evidence that demonstrates that the contribution of synthesis and breakdown to changes in protein abundance induced by REX differ on a protein-by-protein basis. We also highlight the importance of the degradation of individual muscle proteins after exercise in human skeletal muscle.-Camera, D. M., Burniston, J. G., Pogson, M. A., Smiles, W. J., Hawley, J. A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise. © FASEB.

Supplementary Material for: Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

KAUST Repository

Phelan, Jody; Coll, Francesc; McNerney, Ruth; Ascher, David; Pires, Douglas; Furnham, Nick; Coeck, Nele; Hill-Cawthorne, Grant; Nair, Mridul; Mallard, Kim; Ramsay, Andrew; Campino, Susana; Hibberd, Martin; Pain, Arnab; Rigouts, Leen; Clark, Taane

2016-01-01

Abstract Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure

Expression and localization of p-glycoprotein, multidrug resistance protein 4, and breast cancer resistance protein in the female lower genital tract of human and pigtailed macaque.

Science.gov (United States)

Zhou, Tian; Hu, Minlu; Pearlman, Andrew; Patton, Dorothy; Rohan, Lisa

2014-11-01

Antiretroviral drug absorption and disposition in cervicovaginal tissue is important for the effectiveness of vaginally or orally administered drug products in preexposure prophylaxis (PrEP) of HIV-1 sexual transmission to women. Therefore, it is imperative to understand critical determinants of cervicovaginal tissue pharmacokinetics. This study aimed to examine the mRNA expression and protein localization of three efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated protein 4 (MRP4), and breast cancer resistance protein (BCRP), in the lower genital tract of premenopausal women and pigtailed macaques. Along the human lower genital tract, the three transporters were moderately to highly expressed compared to colorectal tissue and liver, as revealed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). In a given genital tract segment, the transporter with the highest expression level was either BCRP or P-gp, while MRP4 was always expressed at the lowest level among the three transporters tested. The immunohistochemical staining showed that P-gp and MRP4 were localized in multiple cell types including epithelial cells and vascular endothelial cells. BCRP was predominantly localized in the vascular endothelial cells. Differences in transporter mRNA level and localization were observed among endocervix, ectocervix, and vagina. Compared to human tissues, the macaque cervicovaginal tissues displayed comparable expression and localization patterns of the three transporters, although subtle differences were observed between the two species. The role of these cervicovaginal transporters in drug absorption and disposition warrants further studies. The resemblance between human and pigtailed macaque in transporter expression and localization suggests the utility of the macaque model in the studies of human cervicovaginal transporters.

Multidrug Resistance Protein-4 Influences Aspirin Toxicity in Human Cell Line

Directory of Open Access Journals (Sweden)

Isabella Massimi

2015-01-01

Full Text Available Overexpression of efflux transporters, in human cells, is a mechanism of resistance to drug and also to chemotherapy. We found that multidrug resistance protein-4 (MRP4 overexpression has a role in reducing aspirin action in patients after bypass surgery and, very recently, we found that aspirin enhances platelet MRP4 levels through peroxisome proliferator activated receptor-α (PPARα. In the present paper, we verified whether exposure of human embryonic kidney-293 cells (Hek-293 to aspirin modifies MRP4 gene expression and its correlation with drug elimination and cell toxicity. We first investigated the effect of high-dose aspirin in Hek-293 and we showed that aspirin is able to increase cell toxicity dose-dependently. Furthermore, aspirin effects, induced at low dose, already enhance MRP4 gene expression. Based on these findings, we compared cell viability in Hek-293, after high-dose aspirin treatment, in MRP4 overexpressing cells, either after aspirin pretreatment or in MRP4 transfected cells; in both cases, a decrease of selective aspirin cell growth inhibition was observed, in comparison with the control cultures. Altogether, these data suggest that exposing cells to low nontoxic aspirin dosages can induce gene expression alterations that may lead to the efflux transporter protein overexpression, thus increasing cellular detoxification of aspirin.

The effects of a high protein diet on indices of health and body composition--a crossover trial in resistance-trained men.

Science.gov (United States)

Antonio, Jose; Ellerbroek, Anya; Silver, Tobin; Vargas, Leonel; Peacock, Corey

2016-01-01

Eight weeks of a high protein diet (>3 g/kg/day) coupled with a periodized heavy resistance training program has been shown to positively affect body composition with no deleterious effects on health. Using a randomized, crossover design, resistance-trained male subjects underwent a 16-week intervention (i.e., two 8-week periods) in which they consumed either their normal (i.e., habitual) or a higher protein diet (>3 g/kg/day). Thus, the purpose of this study was to ascertain if significantly increasing protein intake would affect clinical markers of health (i.e., lipids, kidney function, etc.) as well as performance and body composition in young males with extensive resistance training experience. Twelve healthy resistance-trained men volunteered for this study (mean ± SD: age 25.9 ± 3.7 years; height 178.0 ± 8.5 cm; years of resistance training experience 7.6 ± 3.6) with 11 subjects completing most of the assessments. In a randomized crossover trial, subjects were tested at baseline and after two 8-week treatment periods (i.e., habitual [normal] diet and high protein diet) for body composition, measures of health (i.e., blood lipids, comprehensive metabolic panel) and performance. Each subject maintained a food diary for the 16-week treatment period (i.e., 8 weeks on their normal or habitual diet and 8 weeks on a high protein diet). Each subject provided a food diary of two weekdays and one weekend day per week. In addition, subjects kept a diary of their training regimen that was used to calculate total work performed. During the normal and high protein phase of the treatment period, subjects consumed 2.6 ± 0.8 and 3.3 ± 0.8 g/kg/day of dietary protein, respectively. The mean protein intake over the 4-month period was 2.9 ± 0.9 g/kg/day. The high protein group consumed significantly more calories and protein (p protein group. There were no differences in dietary intake between the groups for any other measure

C-reactive protein, insulin resistance and risk of cardiovascular disease: a population-based study

DEFF Research Database (Denmark)

Hansen, T.W.; Olsen, M.H.; Rasmussen, S.

2008-01-01

BACKGROUND: C-reactive protein (CRP), a marker of inflammation, and insulin resistance (IR), a metabolic disorder, are closely related. CRP and IR have both been identified as significant risk factors of cardiovascular disease (CVD) after adjustment for conventional CVD risk factors...

The 1p-encoded protein stathmin and resistance of malignant gliomas to nitrosoureas.

Science.gov (United States)

Ngo, Teri-T B; Peng, Tien; Liang, Xing-Jie; Akeju, Oluwaseun; Pastorino, Sandra; Zhang, Wei; Kotliarov, Yuri; Zenklusen, Jean C; Fine, Howard A; Maric, Dragan; Wen, Patrick Y; De Girolami, Umberto; Black, Peter McL; Wu, Wells W; Shen, Rong-Fong; Jeffries, Neal O; Kang, Dong-Won; Park, John K

2007-04-18

Malignant gliomas are generally resistant to all conventional therapies. Notable exceptions are anaplastic oligodendrogliomas with loss of heterozygosity on chromosome 1p (1p+/-). Patients with 1p+/- anaplastic oligodendroglioma frequently respond to procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and vincristine. Because the underlying biologic basis for this clinical finding is unclear, we evaluated differentially expressed 1p-encoded proteins in 1p+/- and 1p+/+ malignant glioma cell lines and then examined whether their expression was associated with outcome of patients with anaplastic oligodendroglioma. We used a comparative proteomic screen of A172 (1p+/-) and U251 (1p+/+) malignant glioma cell lines to identify differentially expressed 1p-encoded proteins, including stathmin, a microtubule-associated protein. 1p+/- and 1p+/+ anaplastic oligodendroglioma specimens from 24 patients were assessed for stathmin expression by immunohistochemistry. The relationship between stathmin expression and clinical outcome was assessed with Kaplan-Meier analyses. RNA inhibition and cDNA transfection experiments tested effects of stathmin under- and overexpression, respectively, on the in vitro and in vivo resistance of malignant glioma cells to treatment with nitrosourea. For in vivo resistance studies, 36 mice with intracranial and 16 mice with subcutaneous xenograft tumor implants were used (one tumor per mouse). Flow cytometry was used for cell cycle analysis. Immunoblotting was used to assess protein expression. All statistical tests were two-sided. Decreased stathmin expression in tumors was statistically significantly associated with loss of heterozygosity in 1p (Pnitrosourea-treated mice carrying xenograft tumors. Median survival of mice with stathmin+/- tumors was 95 days (95% CI = 68.7 to 121.3 days) and that of mice with stathmin+/+ tumors was 64 days (95% CI = 58.2 to 69.8 days) (difference = 31 days, 95% CI = 4.1 to 57.9 days; PNitrosoureas induced

Impaired renal secretion of substrates for the multidrug resistance protein 2 in mutant transport-deficient (TR-) rats.

NARCIS (Netherlands)

Masereeuw, R.; Notenboom, S.; Smeets, P.H.E.; Wouterse, A.C.; Russel, F.G.M.

2003-01-01

Previous studies with mutant transport-deficient rats (TR(-)), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is

The effect of taurine and β-alanine supplementation on taurine transporter protein and fatigue resistance in skeletal muscle from mdx mice.

Science.gov (United States)

Horvath, Deanna M; Murphy, Robyn M; Mollica, Janelle P; Hayes, Alan; Goodman, Craig A

2016-11-01

This study investigated the effect of taurine and β-alanine supplementation on muscle function and muscle taurine transporter (TauT) protein expression in mdx mice. Wild-type (WT) and mdx mice (5 months) were supplemented with taurine or β-alanine for 4 weeks, after which in vitro contractile properties, fatigue resistance and force recovery, and the expression of the TauT protein and proteins involved in excitation-contraction (E-C) coupling were examined in fast-twitch muscle. There was no difference in basal TauT protein expression or basal taurine content between mdx than WT muscle. Supplementation with taurine and β-alanine increased and reduced taurine content, respectively, in muscle from WT and mdx mice but had no effect of TauT protein. Taurine supplementation reduced body and muscle mass, and enhanced fatigue resistance and force recovery in mdx muscle. β-Alanine supplementation enhanced fatigue resistance in WT and mdx muscle. There was no difference in the basal expression of key E-C coupling proteins [ryanodine receptor 1 (RyR1), dihydropyridine receptor (DHPR), sarco(endo)plasmic reticulum Ca 2+ -ATPase 1 (SERCA1) or calsequestrin 1 (CSQ1)] between WT and mdx mice, and the expression of these proteins was not altered by taurine or β-alanine supplementation. These findings suggest that TauT protein expression is relatively insensitive to changes in muscle taurine content in WT and mdx mice, and that taurine and β-alanine supplementation may be viable therapeutic strategies to improve fatigue resistance of dystrophic skeletal muscle.

Protein-resistant polymer coatings obtained by matrix assisted pulsed laser evaporation

Energy Technology Data Exchange (ETDEWEB)

Rusen, L. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Mustaciosu, C. [Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH, Magurele, Bucharest (Romania); Mitu, B.; Filipescu, M.; Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Dinca, V., E-mail: dinali@nipne.ro [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania)

2013-08-01

Adsorption of proteins and polysaccharides is known to facilitate microbial attachment and subsequent formation of biofilm on surfaces that ultimately results in its biofouling. Therefore, protein repellent modified surfaces are necessary to block the irreversible attachment of microorganisms. Within this context, the feasibility of using the Poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether (PEG-block-PCL Me) copolymer as potential protein-resistant coating was explored in this work. The films were deposited using Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique that allows good control of composition, thickness and homogeneity. The chemical and morphological characteristics of the films were examined using Fourier Transform Infrared Spectroscopy (FTIR), contact angle measurements and Atomic Force Microscopy (AFM). The FTIR data demonstrates that the functional groups in the MAPLE-deposited films remain intact, especially for fluences below 0.5 J cm{sup −2}. Optical Microscopy and AFM images show that the homogeneity and the roughness of the coatings are related to both laser parameters (fluence, number of pulses) and target composition. Protein adsorption tests were performed on the PEG-block-PCL Me copolymer coated glass and on bare glass surface as a control. The results show that the presence of copolymer as coating significantly reduces the adsorption of proteins.

RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

Science.gov (United States)

Subauste, M Cecilia; Ventura-Holman, Tereza; Du, Liqin; Subauste, Jose S; Chan, Shing-Leng; Yu, Victor C; Maher, Joseph F

2009-12-01

Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

Branched-Chain Amino Acid Ingestion Stimulates Muscle Myofibrillar Protein Synthesis following Resistance Exercise in Humans

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Sarah R. Jackman

2017-06-01

Full Text Available The ingestion of intact protein or essential amino acids (EAA stimulates mechanistic target of rapamycin complex-1 (mTORC1 signaling and muscle protein synthesis (MPS following resistance exercise. The purpose of this study was to investigate the response of myofibrillar-MPS to ingestion of branched-chain amino acids (BCAAs only (i.e., without concurrent ingestion of other EAA, intact protein, or other macronutrients following resistance exercise in humans. Ten young (20.1 ± 1.3 years, resistance-trained men completed two trials, ingesting either 5.6 g BCAA or a placebo (PLA drink immediately after resistance exercise. Myofibrillar-MPS was measured during exercise recovery with a primed, constant infusion of L-[ring13C6] phenylalanine and collection of muscle biopsies pre and 4 h-post drink ingestion. Blood samples were collected at time-points before and after drink ingestion. Western blotting was used to measure the phosphorylation status of mTORC1 signaling proteins in biopsies collected pre, 1-, and 4 h-post drink. The percentage increase from baseline in plasma leucine (300 ± 96%, isoleucine (300 ± 88%, and valine (144 ± 59% concentrations peaked 0.5 h-post drink in BCAA. A greater phosphorylation status of S6K1Thr389 (P = 0.017 and PRAS40 (P = 0.037 was observed in BCAA than PLA at 1 h-post drink ingestion. Myofibrillar-MPS was 22% higher (P = 0.012 in BCAA (0.110 ± 0.009%/h than PLA (0.090 ± 0.006%/h. Phenylalanine Ra was ~6% lower in BCAA (18.00 ± 4.31 μmol·kgBM−1 than PLA (21.75 ± 4.89 μmol·kgBM−1; P = 0.028 after drink ingestion. We conclude that ingesting BCAAs alone increases the post-exercise stimulation of myofibrillar-MPS and phosphorylation status mTORC1 signaling.

Whey protein hydrolysate augments tendon and muscle hypertrophy independent of resistance exercise contraction mode.

Science.gov (United States)

Farup, J; Rahbek, S K; Vendelbo, M H; Matzon, A; Hindhede, J; Bejder, A; Ringgard, S; Vissing, K

2014-10-01

In a comparative study, we investigated the effects of maximal eccentric or concentric resistance training combined with whey protein or placebo on muscle and tendon hypertrophy. 22 subjects were allocated into either a high-leucine whey protein hydrolysate + carbohydrate group (WHD) or a carbohydrate group (PLA). Subjects completed 12 weeks maximal knee extensor training with one leg using eccentric contractions and the other using concentric contractions. Before and after training cross-sectional area (CSA) of m. quadriceps and patellar tendon CSA was quantified with magnetic resonance imaging and a isometric strength test was used to assess maximal voluntary contraction (MVC) and rate of force development (RFD). Quadriceps CSA increased by 7.3 ± 1.0% (P tendon CSA increased by 14.9 ± 3.1% (P effect of contraction mode. MVC and RFD increased by 15.6 ± 3.5% (P effects. In conclusion, high-leucine whey protein hydrolysate augments muscle and tendon hypertrophy following 12 weeks of resistance training - irrespective of contraction mode. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Resistance to the peptidyl transferase inhibitor tiamulin caused by mutation of ribosomal protein l3.

Science.gov (United States)

Bøsling, Jacob; Poulsen, Susan M; Vester, Birte; Long, Katherine S

2003-09-01

The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.

Supplementary Material for: Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

KAUST Repository

Phelan, Jody

2016-01-01

Abstract Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel

Ultraviolet light and ozone stimulate accumulation of salicylic acid, pathogenesis-related proteins and virus resistance in tobacco

International Nuclear Information System (INIS)

Yalpani, N.; Enyedi, A.J.; León, J.; Raskin, I.

1994-01-01

In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 μg·g −1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance. (author)

Up-regulation of DNA-dependent protein kinase correlates with radiation resistance in oral squamous cell carcinoma

International Nuclear Information System (INIS)

Shintani, Satoru; Mihara, Mariko; Li, Chunnan; Nakahara Yuuji; Hino, Satoshi; Nakashiro, Koh-ichi; Hamakawa, Hiroyuki

2003-01-01

DNA-PK is a nuclear protein with serine/threonine kinase activity and forms a complex consisting of the DNA-PKcs and a heterodimer of Ku70 and Ku80 proteins. Recent laboratory experiments have demonstrated that the DNA-PK complex formation is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation. In this study, we evaluated the relationship between expression levels of DNA-PKcs, Ku70 and Ku80 proteins and radiation sensitivity in oral squamous cell carcinoma (OSCC) cell lines and in OSCC patients treated with preoperative radiation therapy. The OSCC cell lines greatly differed in their response to irradiation, as assessed by a standard colony formation assay. However, the expression levels of the DNA-PK complex proteins were all similar, and there was no association between the magnitude of their expression and the tumor radiation sensitivity. Expression of DNA-PK complex proteins increased after radiation treatment, and the increased values correlated with the tumor radiation resistance. Expression of DNA-PKcs and Ku70 after irradiation was increased in the surviving cells of OSCC tissues irradiated preoperatively. These results suggest that up-regulation of DNA-PK complex protein, especially DNA-PKcs, after radiation treatment correlates to radiation resistance. DNA-PKcs might be a molecular target for a novel radiation sensitization therapy of OSCC. (author)

The SNARE protein SNAP23 and the SNARE-interacting protein Munc18c in human skeletal muscle are implicated in insulin resistance/type 2 diabetes

DEFF Research Database (Denmark)

Boström, Pontus; Andersson, Linda; Vind, Birgitte

2010-01-01

/cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS: We have translated our previous in vitro results......OBJECTIVE: Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known...... association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS: We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control...

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

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Jing Dan

2017-03-01

Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in

A Novel Peptide from Soybean Protein Isolate Significantly Enhances Resistance of the Organism under Oxidative Stress.

Directory of Open Access Journals (Sweden)

Heran Ma

Full Text Available Recent studies have indicated that protein hydrolysates have broad biological effects. In the current study we describe a novel antioxidative peptide, FDPAL, from soybean protein isolate (SPI. The aim of this study was to purify and characterize an antioxidative peptide from SPI and determine its antioxidative mechanism. LC-MS/MS was used to isolate and identify the peptide from SPI. The sequence of the peptide was determined to be Phe-Asp-Pro-Ala-Leu (FDPAL, 561 Da. FDPAL can cause significant enhancement of resistance to oxidative stress both in cells as well as simple organisms. In Caenorhabditis elegans (C. elegans, FDPAL can up-regulate the expression of certain genes associated with resistance. The antioxidant activity of this peptide can be attributed to the presence of a specific amino acid sequence. Results from our work suggest that FDPAL can facilitate potential applications of proteins carrying this sequence in the nutraceutical, bioactive material and clinical medicine areas, as well as in cosmetics and health care products.

Poly(l-glutamic acid)-g-poly(ethylene glycol) external layer in polyelectrolyte multilayer films: Characterization and resistance to serum protein adsorption.

Science.gov (United States)

Szczepanowicz, Krzysztof; Kruk, Tomasz; Świątek, Wiktoria; Bouzga, Aud M; Simon, Christian R; Warszyński, Piotr

2018-06-01

Formation of protein-resistant surfaces is a major challenge in the design of novel biomaterials and an important strategy to prevent protein adsorption is the formation of protein-resistant coatings. It can be achieved by proper modification of surfaces, e.g., by immobilization of hydrophilic polymers such as poly(ethylene glycol) (PEG). An appropriate method to immobilize PEG at charged surfaces is the adsorption of copolymers with PEG chains grafted onto polyelectrolyte backbone. The growing interest in the use of polyelectrolyte multilayer coatings in biomedical applications to improve biocompatibility and/or to prepare coating with antiadhesive properties has been the main reason for these studies. Therefore the aim was to produce protein resistant polyelectrolyte multilayer films. They were formed via the layer-by-layer approach, while their pegylation by the deposition of pegylated polyanion, PGA-g-PEG, as an external layer. The influence of PEG chain length and grafting density of PGA-g-PEG copolymers on the protein antiadhesive properties of pegylated polyelectrolyte multilayer films was investigated. To monitor the formation of pegylated and non-pegylated multilayer films, adsorption of the following proteins: HSA, Fibrinogen, and FBS were measured by quartz crystal microbalance (QCM - D). We found that protein adsorption onto all pegylated polyelectrolyte multilayers was significantly reduced in comparison to non-pegylated ones. Long-term performance tests confirmed the stability and the durability of the protein resistant properties of the pegylated multilayers. Antiadhesive properties of tested surfaces pegylated by PGA-g-PEG were compared to the available data for pegylated polycation PLL-g-PEG. Copyright © 2018 Elsevier B.V. All rights reserved.

One-year effectiveness of two hypocaloric diets with different protein/carbohydrate ratios in weight loss and insulin resistance.

Science.gov (United States)

Calleja Fernández, A; Vidal Casariego, A; Cano Rodríguez, I; Ballesteros Pomar, Ma D

2012-01-01

The maintenance of weight loss may be influenced by the distribution of macronutrients in the diet and insulin sensitivity. The objective of the study was to evaluate the longterm effect of two hypocaloric diets with different protein/carbohydrate ratios in overweight and obese individuals either with insulin resistance (IR) or without insulin resistance (IS). Prospective, randomized, clinical intervention study. Forty patients were classified as IR/IS after a 75 g oral glucose tolerance test and then randomized to a diet with either 40% carbohydrate/30% protein/30% fat (diet A) or 55% carbohydrate/15% protein/30% fat (diet B). After one year of follow-up there was no difference in weight loss between diets A and B in each group, but the IS group maintained weight loss better than the IR group [-5.7 (3.9) vs. -0.6 (4.1); P = 0.04]. No differences were found in either Homeostasis Model Assessment (HOMA) or other metabolic glucose parameters except lower insulin at 120 minutes with diet A [21.40 (8.30) vs. 71.40 (17.11); P = 0.02]. The hypocaloric diets with different protein/carbohydrate ratios produced similar changes in weight. Insulin resistance may play a negative role in maintaining weight loss.

XA23 is an executor R protein and confers broad-spectrum disease resistance in rice.

Science.gov (United States)

Wang, Chunlian; Zhang, Xiaoping; Fan, Yinglun; Gao, Ying; Zhu, Qinlong; Zheng, Chongke; Qin, Tengfei; Li, Yanqiang; Che, Jinying; Zhang, Mingwei; Yang, Bing; Liu, Yaoguang; Zhao, Kaijun

2014-11-09

The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE) associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from the wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113-amino acid protein that shares 50% identity to the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23, but differs in promoter region by lacking the TALE binding-element (EBE) for AvrXa23. XA23 can trigger strong hypersensitive response in rice, tobacco and tomato. Our results provide the first evidence that plant genomes have an executor R gene family in which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in pathogen. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells.

Science.gov (United States)

Guerriero, Luana; Palmieri, Giuseppe; De Marco, Margot; Cossu, Antonio; Remondelli, Paolo; Capunzo, Mario; Turco, Maria Caterina; Rosati, Alessandra

2017-10-06

BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule. Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways. Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design.

Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.

Science.gov (United States)

Murphy, Caoileann H; Shankaran, Mahalakshmi; Churchward-Venne, Tyler A; Mitchell, Cameron J; Kolar, Nathan M; Burke, Louise M; Hawley, John A; Kassis, Amira; Karagounis, Leonidas G; Li, Kelvin; King, Chelsea; Hellerstein, Marc; Phillips, Stuart M

2018-06-01

Strategies to enhance the loss of fat while preserving muscle mass during energy restriction are of great importance to prevent sarcopenia in overweight older adults. We show for the first time that the integrated rate of synthesis of numerous individual contractile, cytosolic and mitochondrial skeletal muscle proteins was increased by resistance training (RT) and unaffected by dietary protein intake pattern during energy restriction in free-living, obese older men. We observed a correlation between the synthetic rates of skeletal muscle-derived proteins obtained in serum (creatine kinase M-type, carbonic anhydrase 3) and the synthetic rates of proteins obtained via muscle sampling; and that the synthesis rates of these proteins in serum revealed the stimulatory effects of RT. These results have ramifications for understanding the influence of RT on skeletal muscle and are consistent with the role of RT in maintaining muscle protein synthesis and potentially supporting muscle mass preservation during weight loss. We determined how the pattern of protein intake and resistance training (RT) influenced longer-term (2 weeks) integrated myofibrillar protein synthesis (MyoPS) during energy restriction (ER). MyoPS and proteome kinetics were measured during 2 weeks of ER alone and 2 weeks of ER plus RT (ER + RT) in overweight/obese older men. Participants were randomized to consume dietary protein in a balanced (BAL: 25% daily protein per meal × 4 meals) or skewed (SKEW: 7:17:72:4% daily protein per meal) pattern (n = 10 per group). Participants ingested deuterated water during the consecutive 2-week periods, and skeletal muscle biopsies and serum were obtained at the beginning and conclusion of ER and ER + RT. Bulk MyoPS (i.e. synthesis of the myofibrillar protein sub-fraction) and the synthetic rates of numerous individual skeletal muscle proteins were quantified. Bulk MyoPS was not affected by protein distribution during ER or ER + RT (ER: BAL = 1.24�

Extracellular proteins: Novel key components of metal resistance in cyanobacteria?

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Joaquin eGiner-Lamia

2016-06-01

Full Text Available Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias towards the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria.

Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells.

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Mansilla, Sylvia; Rojas, Marta; Bataller, Marc; Priebe, Waldemar; Portugal, José

2007-04-01

Multidrug-resistance protein 1 (MRP-1) confers resistance to a number of clinically important chemotherapeutic agents. The promoter of the mrp-1 gene contains an Sp1-binding site, which we targeted using the antitumor bis-anthracycline WP631. When MCF-7/VP breast cancer cells, which overexpress MRP-1 protein, were incubated with WP631 the expression of the multidrug-resistance protein gene decreased. Conversely, doxorubicin did not alter mrp-1 gene expression. The inhibition of gene expression was followed by a decrease in the activity of the MRP-1 protein. The IC(75) for WP631 (drug concentration required to inhibit cell growth by 75%) circumvented the drug-efflux pump, without addition of resistant modifiers. After treatment with WP631, MCF-7/VP cells were committed to die after entering mitosis (mitotic catastrophe), while treatment with doxorubicin did not affect cell growth. This is the first report on an antitumor drug molecule inhibiting the mrp-1 gene directly, rather than being simply a poor substrate for the transporter-mediated efflux. However, both situations appeared to coexist, thereby a superior cytotoxic effect was attained. Ours results suggest that WP631 offers great potential for the clinical treatment of tumors displaying a multidrug-resistance phenotype.

Ribosomal protein L3 mutations are associated with cfr-mediated linezolid resistance in clinical isolates of Staphylococcus cohnii.

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Xu, Hongtao; Tian, Rui; Li, Yanming; Chen, Dongke; Liu, Yalin; Hu, Yunjian; Xiao, Fei

2015-06-01

From June, 2012 to November, 2013 five linezolid-resistant Staphylococcus cohnii isolates were identified in our hospital in Beijing, China. The investigation of the resistance mechanisms confirmed that the cfr-carrying plasmids were the main cause of linezolid resistance in those clinical isolates. Moreover, all the five isolates had ribosomal protein L3 mutations, which had different coordinate effect on cfr-mediated linezolid resistance directly through the substitution of serine 158 by phenylalanine or tyrosine in L3 protein. In this study, two types of plasmids (p432, p438) (Accession No. KM114207) were found, which share high sequence identity with previously reported cfr-carrying pRM01 and pMHZ plasmids originated from northern and southern China, showing wide regional dissemination in China. The stability of linezolid resistance was studied by passaging single colonies serially on antibiotic-free blood medium, which showed that the susceptible derivatives emerged until the passages 39-42 with the elimination of cfr-carrying plasmid. Thus the high stability of this plasmid may pose a risk for the transmission among patients or even cause an outbreak in clinical settings.

Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet (Eleusine coracana).

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Panwar, Preety; Jha, Anand Kumar; Pandey, P K; Gupta, Arun K; Kumar, Anil

2011-06-01

Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05(504,) NBS-09(711), NBS-07(688), NBS-03(509) and EST-SSR-04(241)) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.

Defects in mitochondrial fission protein dynamin-related protein 1 are linked to apoptotic resistance and autophagy in a lung cancer model.

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Kelly Jean Thomas

Full Text Available Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549 cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype-mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1. A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy. A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.

Salivary protein polymorphisms and risk of dental caries: a systematic review.

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Lips, Andrea; Antunes, Leonardo Santos; Antunes, Lívia Azeredo; Pintor, Andrea Vaz Braga; Santos, Diana Amado Baptista Dos; Bachinski, Rober; Küchler, Erika Calvano; Alves, Gutemberg Gomes

2017-06-05

Dental caries is an oral pathology associated with both lifestyle and genetic factors. The caries process can be influenced by salivary composition, which includes ions and proteins. Studies have described associations between salivary protein polymorphisms and dental caries experience, while others have shown no association with salivary proteins genetic variability. The aim of this study is to assess the influence of salivary protein polymorphisms on the risk of dental caries by means of a systematic review of the current literature. An electronic search was performed in PubMed, Scopus, and Virtual Health Library. The following search terms were used: "dental caries susceptibility," "dental caries," "polymorphism, genetics," "saliva," "proteins," and "peptides." Related MeSH headings and free terms were included. The inclusion criteria comprised clinical investigations of subjects with and without caries. After application of these eligibility criteria, the selected articles were qualified by assessing their methodological quality. Initially, 338 articles were identified from the electronic databases after exclusion of duplicates. Exclusion criteria eliminated 322 articles, and 16 remained for evaluation. Eleven articles found a consistent association between salivary protein polymorphisms and risk of dental caries, for proteins related to antimicrobial activity (beta defensin 1 and lysozyme-like protein), pH control (carbonic anhydrase VI), and bacterial colonization/adhesion (lactotransferrin, mucin, and proline-rich protein Db). This systematic review demonstrated an association between genetic polymorphisms and risk of dental caries for most of the salivary proteins.

Low-load high volume resistance exercise stimulates muscle protein synthesis more than high-load low volume resistance exercise in young men.

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Nicholas A Burd

Full Text Available BACKGROUND: We aimed to determine the effect of resistance exercise intensity (%1 repetition maximum-1RM and volume on muscle protein synthesis, anabolic signaling, and myogenic gene expression. METHODOLOGY/PRINCIPAL FINDINGS: Fifteen men (21+/-1 years; BMI=24.1+/-0.8 kg/m2 performed 4 sets of unilateral leg extension exercise at different exercise loads and/or volumes: 90% of repetition maximum (1RM until volitional failure (90FAIL, 30% 1RM work-matched to 90%FAIL (30WM, or 30% 1RM performed until volitional failure (30FAIL. Infusion of [ring-13C6] phenylalanine with biopsies was used to measure rates of mixed (MIX, myofibrillar (MYO, and sarcoplasmic (SARC protein synthesis at rest, and 4 h and 24 h after exercise. Exercise at 30WM induced a significant increase above rest in MIX (121% and MYO (87% protein synthesis at 4 h post-exercise and but at 24 h in the MIX only. The increase in the rate of protein synthesis in MIX and MYO at 4 h post-exercise with 90FAIL and 30FAIL was greater than 30WM, with no difference between these conditions; however, MYO remained elevated (199% above rest at 24 h only in 30FAIL. There was a significant increase in AktSer473 at 24h in all conditions (P=0.023 and mTORSer2448 phosphorylation at 4 h post-exercise (P=0.025. Phosporylation of Erk1/2Tyr202/204, p70S6KThr389, and 4E-BP1Thr37/46 increased significantly (P<0.05 only in the 30FAIL condition at 4 h post-exercise, whereas, 4E-BP1Thr37/46 phosphorylation was greater 24 h after exercise than at rest in both 90FAIL (237% and 30FAIL (312% conditions. Pax7 mRNA expression increased at 24 h post-exercise (P=0.02 regardless of condition. The mRNA expression of MyoD and myogenin were consistently elevated in the 30FAIL condition. CONCLUSIONS/SIGNIFICANCE: These results suggest that low-load high volume resistance exercise is more effective in inducing acute muscle anabolism than high-load low volume or work matched resistance exercise modes.

Whey and casein labelled with L-[1-13C]-leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion

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Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

2011-01-01

to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass......), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments......, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after...

Dietary sardine protein lowers insulin resistance, leptin and TNF-α and beneficially affects adipose tissue oxidative stress in rats with fructose-induced metabolic syndrome.

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Madani, Zohra; Louchami, Karim; Sener, Abdullah; Malaisse, Willy J; Ait Yahia, Dalila

2012-02-01

The present study aims at exploring the effects of sardine protein on insulin resistance, plasma lipid profile, as well as oxidative and inflammatory status in rats with fructose-induced metabolic syndrome. Rats were fed sardine protein (S) or casein (C) diets supplemented or not with high-fructose (HF) for 2 months. Rats fed the HF diets had greater body weight and adiposity and lower food intake as compared to control rats. Increased plasma glucose, insulin, HbA1C, triacylglycerols, free fatty acids and impaired glucose tolerance and insulin resistance was observed in HF-fed rats. Moreover, a decline in adipose tissues antioxidant status and a rise in lipid peroxidation and plasma TNF-α and fibrinogen were noted. Rats fed sardine protein diets exhibited lower food intake and fat mass than those fed casein diets. Sardine protein diets diminished plasma insulin and insulin resistance. Plasma triacylglycerol and free fatty acids were also lower, while those of α-tocopherol, taurine and calcium were enhanced as compared to casein diets. Moreover, S-HF diet significantly decreased plasma glucose and HbA1C. Sardine protein consumption lowered hydroperoxide levels in perirenal and brown adipose tissues. The S-HF diet, as compared to C-HF diet decreased epididymal hydroperoxides. Feeding sardine protein diets decreased brown adipose tissue carbonyls and increased glutathione peroxidase activity. Perirenal and epididymal superoxide dismutase and catalase activities and brown catalase activity were significantly greater in S-HF group than in C-HF group. Sardine protein diets also prevented hyperleptinemia and reduced inflammatory status in comparison with rats fed casein diets. Taken together, these results support the beneficial effect of sardine protein in fructose-induced metabolic syndrome on such variables as hyperglycemia, insulin resistance, hyperlipidemia and oxidative and inflammatory status, suggesting the possible use of sardine protein as a protective

Resistant starch and protein intake enhances fat oxidation and feelings of fullness in lean and overweight/obese women

DEFF Research Database (Denmark)

Gentile, Christopher L; Ward, Emery; Holst, Jens Juul

2015-01-01

and overweight/obese women. METHODS: Women of varying levels of adiposity consumed one of four pancake test meals in a single-blind, randomized crossover design: 1) waxy maize (control) starch (WMS); 2) waxy maize starch and whey protein (WMS+WP); 3) resistant starch (RS); or 4) RS and whey protein (RS...

Challenging metastatic breast cancer with the natural defensin PvD1.

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Figueira, Tiago N; Oliveira, Filipa D; Almeida, Inês; Mello, Érica O; Gomes, Valdirene M; Castanho, Miguel A R B; Gaspar, Diana

2017-11-09

Metastatic breast cancer is a very serious life threatening condition that poses many challenges for the pharmaceutical development of effective chemotherapeutics. As the therapeutics targeted to the localized masses in breast improve, metastatic lesions in the brain slowly increase in their incidence compromising successful treatment outcomes overall. The blood-brain-barrier (BBB) is one important obstacle for the management of breast cancer brain metastases. New therapeutic approaches are in demand for overcoming the BBB's breaching by breast tumor cells. In this work we demonstrate the potential dual role of a natural antimicrobial plant defensin, PvD 1 : it interferes with the formation of solid tumors in the breast and concomitantly controls adhesion of breast cancer cells to human brain endothelial cells. We have used a combination of techniques that probe PvD 1 's effect at the single cell level and reveal that this peptide can effectively damage breast tumor cells, leaving healthy breast and brain cells unaffected. Results suggest that PvD1 quickly internalizes in cancer cells but remains located in the membrane of normal cells with no significant damage to its structure and biomechanical properties. These interactions in turn modulate cell adhesiveness between tumor and BBB cells. PvD 1 is a potential template for the design of innovative pharmacological approaches for metastatic breast cancer treatment: the manipulation of the biomechanical properties of tumor cells that ultimately prevent their attachment to the BBB.

Leucine supplementation improves acquired growth hormone resistance in rats with protein-energy malnutrition.

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Gao, Xuejin; Tian, Feng; Wang, Xinying; Zhao, Jie; Wan, Xiao; Zhang, Li; Wu, Chao; Li, Ning; Li, Jieshou

2015-01-01

Protein-energy malnutrition (PEM) can lead to growth hormone (GH) resistance. Leucine supplementation diets have been shown to increase protein synthesis in muscles. Our study aimed at investigating if long-term leucine supplementation could modulate GH-insulin-like growth factor (IGF)-1 system function and mammalian target of rapamycin (mTOR)-related signal transduction in skeletal muscles in a rat model of severe malnutrition. Male Sprague-Dawley rats (n = 50; weight, 302 ± 5 g) were divided into 5 treatment groups, including 2 control groups (a normal control group that was fed chow and ad libitum water [CON, n = 10] and a malnourished control group [MC, n = 10] that was fed a 50% chow diet). After undergoing a weight loss stage for 4 weeks, rats received either the chow diet (MC-CON, n = 10), the chow diet supplemented with low-dose leucine (MC-L, n = 10), or the chow diet supplemented with high-dose leucine (MC-H, n = 10) for 2 weeks. The muscle masses of the gastrocnemius, soleus, and extensor digitorum longus were significantly reduced in the MC group. Re-feeding increased muscle mass, especially in the MC-L and MC-H groups. In the MC group, serum IGF-1, IGF-binding protein (IGFBP)-3, and hepatic growth hormone receptor (GHR) levels were significantly decreased and phosphorylation of the downstream anabolic signaling effectors protein kinase B (Akt), mTOR, and ribosomal protein S6 kinase 1 (S6K1) were significantly lower than in other groups. However, serum IGF-1 and IGF binding protein (IGFBP)-3 concentrations and hepatic growth hormone receptor (GHR) levels were significantly higher in the MC-L and MC-H groups than in the MC-CON group, and serum IGFBP-1 levels was significantly reduced in the MC-L and MC-H groups. These changes were consistent with those observed for hepatic mRNA expression levels. Phosphorylation of the downstream anabolic signaling effectors Akt, mTOR, and S6K1 were also significantly higher in the MC-L and MC-H groups than in the MC

Effects of Genetically Modified Milk Containing Human Beta-Defensin-3 on Gastrointestinal Health of Mice.

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Xin Chen

Full Text Available This study was performed to investigate the effects of genetically modified (GM milk containing human beta-defensin-3 (HBD3 on mice by a 90-day feeding study. The examined parameters included the digestibility of GM milk, general physical examination, gastric emptying function, intestinal permeability, intestinal microflora composition of mice, and the possibility of horizontal gene transfer (HGT. The emphasis was placed on the effects on gastrointestinal (GI tract due to the fact that GI tract was the first site contacting with food and played crucial roles in metabolic reactions, nutrition absorption and immunity regulation in the host. However, the traditional methods for analyzing the potential toxicological risk of GM product pay little attention on GI health. In this study, the results showed GM milk was easy to be digested in simulated gastric fluid, and it did not have adverse effects on general and GI health compared to conventional milk. And there is little possibility of HGT. This study may enrich the safety assessment of GM product on GI health.

Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli

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Wernimont, A.K.; Huffman, D.L.; Finney, L.A.; Demeler, B.; O' Halloran, T.V.; Rosenzweig, A.C.

2010-03-08

PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli. Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences. Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known. Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins. The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded barrel with the methionines unexpectedly housed on a solvent-exposed loop. Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC. In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit. In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface. These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA. Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein. Dimerization is significantly enhanced upon binding Cu(I) with a measured {Delta}({Delta}G{sup o}) {le} -8.0 kJ/mole, suggesting that copper might bind at the dimer interface.

Effect of manuka honey on the expression of universal stress protein A in meticillin-resistant Staphylococcus aureus.

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Jenkins, Rowena; Burton, Neil; Cooper, Rose

2011-04-01

Staphylococcus aureus is an important pathogen that can cause many problems, from impetigo to endocarditis. With its continued resistance to multiple antibiotics, S. aureus remains a serious health threat. Honey has been used to eradicate meticillin-resistant S. aureus (MRSA) strains from wounds, but its mode of action is not yet understood. Proteomics provides a potent group of techniques that can be used to analyse differences in protein expression between untreated bacterial cells and those treated with inhibitory concentrations of manuka honey. In this study, two-dimensional (2D) electrophoresis was combined with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to determine the identities of proteins whose levels of expression were changed at least two-fold following treatment with manuka honey. Protein extracts were obtained from cells grown in tryptone soy broth (with or without manuka honey) by mechanical disruption and were separated on 2D polyacrylamide gels. A protein was isolated in gels prepared from untreated cell extract that was absent from gels made using honey-treated cell extract. Using MALDI-TOF MS, the protein was identified as universal stress protein A (UspA). Downregulation of this protein was confirmed by real-time polymerase chain reaction (PCR), which showed a 16-fold downregulation in honey-treated cells compared with untreated samples. This protein is involved in the stress stamina response and its downregulation could help to explain the inhibition of MRSA by manuka honey. Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Bph32, a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice

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Ren, Juansheng; Gao, Fangyuan; Wu, Xianting; Lu, Xianjun; Zeng, Lihua; Lv, Jianqun; Su, Xiangwen; Luo, Hong; Ren, Guangjun

2016-01-01

An urgent need exists to identify more brown planthopper (Nilaparvata lugens Stål, BPH) resistance genes, which will allow the development of rice varieties with resistance to BPH to counteract the increased incidence of this pest species. Here, using bioinformatics and DNA sequencing approaches, we identified a novel BPH resistance gene, LOC_Os06g03240 (MSU LOCUS ID), from the rice variety Ptb33 in the interval between the markers RM19291 and RM8072 on the short arm of chromosome 6, where a gene for resistance to BPH was mapped by Jirapong Jairin et al. and renamed as “Bph32”. This gene encodes a unique short consensus repeat (SCR) domain protein. Sequence comparison revealed that the Bph32 gene shares 100% sequence identity with its allele in Oryza latifolia. The transgenic introgression of Bph32 into a susceptible rice variety significantly improved resistance to BPH. Expression analysis revealed that Bph32 was highly expressed in the leaf sheaths, where BPH primarily settles and feeds, at 2 and 24 h after BPH infestation, suggesting that Bph32 may inhibit feeding in BPH. Western blotting revealed the presence of Pph (Ptb33) and Tph (TN1) proteins using a Penta-His antibody, and both proteins were insoluble. This study provides information regarding a valuable gene for rice defence against insect pests. PMID:27876888

Bph32, a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice.

Science.gov (United States)

Ren, Juansheng; Gao, Fangyuan; Wu, Xianting; Lu, Xianjun; Zeng, Lihua; Lv, Jianqun; Su, Xiangwen; Luo, Hong; Ren, Guangjun

2016-11-23

An urgent need exists to identify more brown planthopper (Nilaparvata lugens Stål, BPH) resistance genes, which will allow the development of rice varieties with resistance to BPH to counteract the increased incidence of this pest species. Here, using bioinformatics and DNA sequencing approaches, we identified a novel BPH resistance gene, LOC_Os06g03240 (MSU LOCUS ID), from the rice variety Ptb33 in the interval between the markers RM19291 and RM8072 on the short arm of chromosome 6, where a gene for resistance to BPH was mapped by Jirapong Jairin et al. and renamed as "Bph32". This gene encodes a unique short consensus repeat (SCR) domain protein. Sequence comparison revealed that the Bph32 gene shares 100% sequence identity with its allele in Oryza latifolia. The transgenic introgression of Bph32 into a susceptible rice variety significantly improved resistance to BPH. Expression analysis revealed that Bph32 was highly expressed in the leaf sheaths, where BPH primarily settles and feeds, at 2 and 24 h after BPH infestation, suggesting that Bph32 may inhibit feeding in BPH. Western blotting revealed the presence of Pph (Ptb33) and Tph (TN1) proteins using a Penta-His antibody, and both proteins were insoluble. This study provides information regarding a valuable gene for rice defence against insect pests.