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Sample records for agouti-related peptide expression

  1. Expression of melanocortin-4 receptor and agouti-related peptide mRNAs in arcuate nucleus during long term malnutrition of female ovariectomized rats

    PubMed Central

    Sarvestani, Fatemeh Sabet; Tamadon, Amin; Hematzadeh, Aida; Jahanara, Maliheh; Shirazi, Mohammad Reza Jafarzadeh; Moghadam, Ali; Niazi, Ali; Moghiminasr, Reza

    2015-01-01

    Objective: Melanocortin-4 receptor (MC4R) and agouti-related peptide (AgRP) are involved in energy homeostasis in the rat. The aim of the present study was to evaluate the expression of MC4R and AgRP mRNAs in arcuate nucleus (ARC) during long term malnutrition of female ovariectomized rats. Materials and Methods: Ten female ovariectomized rats were divided into two equal groups (n=6) of normal and restricted diet groups. Using real-time PCR, the relative expressions (compared to controls) of MC4R and AgRP mRNAs were compared between both diet groups. Results: The relative expression of MC4R and AgRP mRNA in the ARC of female ovariectomized rats during long term malnutrition was higher than those with normal diet (P<0.05). Conclusion: Changes in the relative expression level of MC4R and AgRP mRNAs during long term malnutrition of rat indicated a stimulatory role of MC4R and AgRP in regulating energy balance in ARC of rat hypothalamus. PMID:25825637

  2. Hypothalamic Agouti-Related Peptide mRNA is Elevated During Natural and Stress-Induced Anorexia.

    PubMed

    Dunn, I C; Wilson, P W; D'Eath, R B; Boswell, T

    2015-09-01

    As part of their natural lives, animals can undergo periods of voluntarily reduced food intake and body weight (i.e. animal anorexias) that are beneficial for survival or breeding, such as during territorial behaviour, hibernation, migration and incubation of eggs. For incubation, a change in the defended level of body weight or 'sliding set point' appears to be involved, although the neural mechanisms reponsible for this are unknown. We investigated how neuropeptide gene expression in the arcuate nucleus of the domestic chicken responded to a 60-70% voluntary reduction in food intake measured both after incubation and after an environmental stressor involving transfer to unfamiliar housing. We hypothesised that gene expression would not change in these circumstances because the reduced food intake and body weight represented a defended level in birds with free access to food. Unexpectedly, we observed increased gene expression of the orexigenic peptide agouti-related peptide (AgRP) in both incubating and transferred animals compared to controls. Also pro-opiomelanocortin (POMC) mRNA was higher in incubating hens and significantly increased 6 days after exposure to the stressor. Conversely expression of neuropeptide Y and cocaine- and amphetamine-regulated transcript gene was unchanged in both experimental situations. We conclude that AgRP expression remains sensitive to the level of energy stores during natural anorexias, which is of adaptive advantage, although its normal orexigenic effects are over-ridden by inhibitory signals. In the case of stress-induced anorexia, increased POMC may contribute to this inhibitory role, whereas, for incubation, reduced feeding may also be associated with increased expression in the hypothalamus of the anorexigenic peptide vasoactive intestinal peptide.

  3. Short-days induce weight loss in Siberian hamsters despite overexpression of the agouti-related peptide gene.

    PubMed

    Jethwa, P H; Warner, A; Fowler, M J; Murphy, M; de Backer, M W; Adan, R A H; Barrett, P; Brameld, J M; Ebling, F J P

    2010-06-01

    Many vertebrates express profound annual cycles of body fattening, although it is not clear whether these represent differential activity of the central pathways known to mediate homeostatic control of food intake and energy expenditure, or whether the recent discovery of a major role for pars tuberalis-ependymal signalling points towards novel mechanisms. We examined this in the Siberian hamster (Phodopus sungorus) by using gene transfection to up-regulate a major orexigenic peptide, agouti-related peptide (AgRP), and then determined whether this increased anabolic drive could prevent the short-day induced winter catabolic state. Infusions of a recombinant adeno-associated virus encoding an AgRP construct into the hypothalamus of hamsters in the long-day obese phase of their seasonal cycle produced a 20% gain in body weight over 6 weeks compared to hamsters receiving a control reporter construct, reflecting a significant increase in food intake and a significant decrease in energy expenditure. However, all hamsters showed a significant, prolonged decrease in body weight when exposed to short photoperiods, despite the hamsters expressing the AgRP construct maintaining a higher food intake and lower energy expenditure relative to the control hamsters. Visualisation of the green fluorescent protein reporter and analysis of AgRP-immunoreactivity confirmed widespread expression of the construct in the hypothalamus, which was maintained for the 21-week duration of the study. In conclusion, the over-expression of AgRP in the hypothalamus produced a profoundly obese state but did not block the seasonal catabolic response, suggesting a separation of rheostatic mechanisms in seasonality from those maintaining homeostasis of energy metabolism.

  4. Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose.

    PubMed

    Hao, Lihong; Sheng, Zhenyu; Potian, Joseph; Deak, Adam; Rohowsky-Kochan, Christine; Routh, Vanessa H

    2016-10-01

    A population of Neuropeptide Y (NPY) neurons which co-express Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) are inhibited at physiological levels of brain glucose and activated when glucose levels decline (e.g. glucose-inhibited or GI neurons). Fasting enhances the activation of NPY/AgRP-GI neurons by low glucose. In the present study we tested the hypothesis that lipopolysaccharide (LPS) inhibits the enhanced activation of NPY/AgRP-GI neurons by low glucose following a fast. Mice which express green fluorescent protein (GFP) on their NPY promoter were used to identify NPY/AgRP neurons. Fasting for 24h and LPS injection decreased blood glucose levels. As we have found previously, fasting increased c-fos expression in NPY/AgRP neurons and increased the activation of NPY/AgRP-GI neurons by decreased glucose. As we predicted, LPS blunted these effects of fasting at the 24h time point. Moreover, the inflammatory cytokine tumor necrosis factor alpha (TNFα) blocked the activation of NPY/AgRP-GI neurons by decreased glucose. These data suggest that LPS and TNFα may alter glucose and energy homeostasis, in part, due to changes in the glucose sensitivity of NPY/AgRP neurons. Interestingly, our findings also suggest that NPY/AgRP-GI neurons use a distinct mechanism to sense changes in extracellular glucose as compared to our previous studies of GI neurons in the adjacent ventromedial hypothalamic nucleus.

  5. Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose.

    PubMed

    Hao, Lihong; Sheng, Zhenyu; Potian, Joseph; Deak, Adam; Rohowsky-Kochan, Christine; Routh, Vanessa H

    2016-10-01

    A population of Neuropeptide Y (NPY) neurons which co-express Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) are inhibited at physiological levels of brain glucose and activated when glucose levels decline (e.g. glucose-inhibited or GI neurons). Fasting enhances the activation of NPY/AgRP-GI neurons by low glucose. In the present study we tested the hypothesis that lipopolysaccharide (LPS) inhibits the enhanced activation of NPY/AgRP-GI neurons by low glucose following a fast. Mice which express green fluorescent protein (GFP) on their NPY promoter were used to identify NPY/AgRP neurons. Fasting for 24h and LPS injection decreased blood glucose levels. As we have found previously, fasting increased c-fos expression in NPY/AgRP neurons and increased the activation of NPY/AgRP-GI neurons by decreased glucose. As we predicted, LPS blunted these effects of fasting at the 24h time point. Moreover, the inflammatory cytokine tumor necrosis factor alpha (TNFα) blocked the activation of NPY/AgRP-GI neurons by decreased glucose. These data suggest that LPS and TNFα may alter glucose and energy homeostasis, in part, due to changes in the glucose sensitivity of NPY/AgRP neurons. Interestingly, our findings also suggest that NPY/AgRP-GI neurons use a distinct mechanism to sense changes in extracellular glucose as compared to our previous studies of GI neurons in the adjacent ventromedial hypothalamic nucleus. PMID:27473896

  6. Hypothalamic agouti-related protein expression is affected by both acute and chronic experience of food restriction and re-feeding in chickens.

    PubMed

    Dunn, I C; Wilson, P W; Smulders, T V; Sandilands, V; D'Eath, R B; Boswell, T

    2013-10-01

    The central melanocortin system is conserved across vertebrates. However, in birds, little is known about how energy balance influences orexigenic agouti-related protein (AGRP) and anorexigenic pro-opiomelanocortin (POMC) expression, despite the fact that commercial food restriction is critical to the efficient production of poultry meat. To enable contrasts to be made, in broiler-breeder chickens, between levels of food restriction, between birds with the same body weight but different feeding experience, and between birds moved from restricted feeding to ad lib. feeding for different periods, five groups of hens were established between 6 and 12 weeks of age with different combinations of food restriction and release from restriction. AGRP and neuropeptide Y expression in the basal hypothalamus was significantly increased by chronic restriction but only AGRP mRNA levels reflected recent feeding experience: hens at the same body weight that had recently been on ad lib. feeding showed lower expression than restricted birds. AGRP expression also distinguished between hens released from restriction to ad lib. feeding for different periods. By contrast, POMC and cocaine- and amphetamine-regulated transcript mRNA levels were not different. These results showed that AGRP mRNA not only reflected differences between a bird's weight and its potential weight or set point, but also discriminated between differing feeding histories of birds at the same body weight. Therefore, AGRP expression potentially provides an integrated measure of food intake experience and an objective tool to assess a bird's perception of satiety in feeding regimes for improved poultry welfare.

  7. Identification of Distant Agouti-Like Sequences and Re-Evaluation of the Evolutionary History of the Agouti-Related Peptide (AgRP)

    PubMed Central

    Västermark, Åke; Krishnan, Arunkumar; Houle, Michael E.; Fredriksson, Robert; Cerdá-Reverter, José Miguel; Schiöth, Helgi B.

    2012-01-01

    The Agouti-like peptides including AgRP, ASIP and the teleost-specific A2 (ASIP2 and AgRP2) peptides have potent and diverse functional roles in feeding, pigmentation and background adaptation mechanisms. There are contradictory theories about the evolution of the Agouti-like peptide family as well the nomenclature. Here we performed comprehensive mining and annotation of vertebrate Agouti-like sequences. We identified A2 sequences from salmon, trout, seabass, cod, cichlid, tilapia, gilt-headed sea bream, Antarctic toothfish, rainbow smelt, common carp, channel catfish and interestingly also in lobe-finned fish. Moreover, we surprisingly found eight novel homologues from the kingdom of arthropods and three from fungi, some sharing the characteristic C-x(6)-C-C motif which are present in the Agouti-like sequences, as well as approximate sequence length (130 amino acids), positioning of the motif sequence and sharing of exon-intron structures that are similar to the other Agouti-like peptides providing further support for the common origin of these sequences. Phylogenetic analysis shows that the AgRP sequences cluster basally in the tree, suggesting that these sequences split from a cluster containing both the ASIP and the A2 sequences. We also used a novel approach to determine the statistical evidence for synteny, a sinusoidal Hough transform pattern recognition technique. Our analysis shows that the teleost AgRP2 resides in a chromosomal region that has synteny with Hsa 8, but we found no convincing synteny between the regions that A2, AgRP and ASIP reside in, which would support that the Agouti-like peptides were formed by whole genome tetraplodization events. Here we suggest that the Agouti-like peptide genes were formed through classical subsequent gene duplications where the AgRP is the most distantly related to the three other members of that group, first splitting from a common ancestor to ASIP and A2, and then later the A2 split from ASIP followed by a

  8. Expression of neuropeptide Y and agouti-related protein mRNA stimulated by glucocorticoids is attenuated via NF-κB p65 under ER stress in mouse hypothalamic cultures.

    PubMed

    Hagimoto, Shigeru; Arima, Hiroshi; Adachi, Koichi; Ito, Yoshihiro; Suga, Hidetaka; Sugimura, Yoshihisa; Goto, Motomitsu; Banno, Ryoichi; Oiso, Yutaka

    2013-10-11

    There are several lines of evidence suggesting that glucocorticoid signaling in the hypothalamus plays an important role in energy balance, and recent studies suggest that endoplasmic reticulum (ER) stress in the hypothalamus could affect signaling related to energy balance. In the present study, we examined the regulation of glucocorticoid signaling under ER stress in mouse hypothalamic organotypic cultures. Incubation of the hypothalamic explants with dexamethasone (DEX) significantly increased expression levels of neuropeptide Y (NPY) and agouti-related protein (AgRP) mRNA, and treatment with thapsigargin (TG), an ER stressor, significantly attenuated DEX-induced NPY and AgRP mRNA expression. TG treatment increased the levels of phospho-NF-κB p65 in hypothalamic cultures, and inhibitors of NF-κB p65 reversed the inhibitory effects of TG on NPY and AgRP expression. Our data thus demonstrated that glucocorticoid-stimulated NPY and AgRP expression was attenuated via NF-κB p65 pathways under ER stress, and suggest crosstalk between ER stress and inflammation in the hypothalamus.

  9. Characterization, tissue distribution and regulation of agouti-related protein (AgRP) in a cyprinid fish (Schizothorax prenanti).

    PubMed

    Wei, RongBin; Yuan, DengYue; Wang, Tao; Zhou, ChaoWei; Lin, FangJun; Chen, Hu; Wu, HongWei; Yang, ShiYong; Wang, Yan; Liu, Ju; Gao, YunDi; Li, ZhiQiong

    2013-09-15

    Agouti-related protein (AgRP) is an important neuropeptide involved in the regulation of feeding in both mammals and fish. In this study, we have cloned the full-length cDNA sequence for AgRP in a cyprinid fish (Schizothorax prenanti). The AgRP gene, encoding 126-amino acids, was strongly expressed in the brain. The AgRP gene was detected in embryos at developmental stages. Further, its mRNA was detectable in unfertilized eggs. An experiment was conducted to determine the expression profile of AgRP during short-term and long-term fasting of the hypothalamus. The expression level of AgRP in unfed fish was significantly increased at 3 and 4h post-fasting than in fed fish but did not affect AgRP mRNA expression after 14 days fasting. Overall, our results suggest that AgRP is a conserved peptide that might be involved in the regulation of short-term feeding and other physiological function in Schizothorax prenanti.

  10. Central infusion of the melanocortin receptor antagonist agouti-related peptide (AgRP(83-132)) prevents cachexia-related symptoms induced by radiation and colon-26 tumors in mice.

    PubMed

    Joppa, M A; Gogas, K R; Foster, A C; Markison, S

    2007-03-01

    Cachexia is a clinical wasting syndrome that occurs in multiple disease states, and is associated with anorexia and a progressive loss of body fat and lean mass. The development of new therapeutics for this disorder is needed due to poor efficacy and multiple side effects of current therapies. The pivotal role played by the central melanocortin system in regulating body weight has made this an attractive target for novel cachexia therapies. The mixed melanocortin receptor antagonist AgRP is an endogenous peptide that induces hyperphagia. Here, we used AgRP(83-132) to investigate the ability of melanocortin antagonism to protect against clinical features of cachexia in two distinct animal models. In an acute model, food intake and body weight gain were reduced in mice exposed to radiation (300 RAD), and delivery of AgRP(83-132) into the lateral cerebral ventricle prevented these effects. In a chronic tumor cachexia model, adult mice were injected subcutaneously with a cell line derived from murine colon-26 adenocarcinoma. Typical of cachexia, tumor-bearing mice progressively reduced body weight and food intake, and gained significantly less muscle mass than controls. Administration of AgRP(83-132) into the lateral ventricles significantly increased body weight and food intake, and changes in muscle mass were similar to the tumor-free control mice. These findings support the idea that antagonism of the central melanocortin system can reduce the negative impact of cachexia and radiation therapy.

  11. Disulfide assignment of the C-terminal cysteine knot of agouti-related protein (AGRP) by direct sequencing analysis.

    PubMed

    Young, Y; Zeni, L; Rosenfeld, R D; Stark, K L; Rohde, M F; Haniu, M

    1999-12-01

    We have assigned the disulfide structure of Md-65 agouti-related protein (Md65-AGRP) using differential reduction and alkylation followed by direct sequencing analysis. The mature human AGRP is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The C-terminal domain, a 48 amino acid peptide named Md65-AGRP, was expressed in Escherichia coil cells and refolded under different conditions from the mature recombinant protein. The disulfide bonds in the cystine knot structure of Md65-AGRP were partially reduced using tris(2-carboxyethyl) phosphine (TCEP) under acidic conditions, followed by alkylation with N-ethylmaleimide (NEM). The procedure generated several isoforms with varying degrees of NEM alkylation. The multiple forms of Md65-AGRP generated by partial reduction and NEM modification were then completely reduced and carboxymethylated to identify unreactive disulfide bonds. Differentially labeled Md65-AGRP were directly sequenced and analyzed by MALDI mass spectrometry. The results confirmed that Md65-AGRP contained the same disulfide structure as that of Md5-AGRP reported previously [Bures, E. J., Hui, J. O., Young, Y. et al. (1998) Biochemistry 37, 12172-12177].

  12. Diurnal rhythm of agouti-related protein and its relation to corticosterone and food intake.

    PubMed

    Lu, Xin-Yun; Shieh, Kun-Ruey; Kabbaj, Mohamed; Barsh, Gregory S; Akil, Huda; Watson, Stanley J

    2002-10-01

    In the present study we examined the diurnal patterns of agouti-related protein (AGRP) and proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus and their relation to circulating glucocorticoids and food intake. Animals were killed at 4-h intervals throughout the 24-h diurnal cycle, and the expression of AGRP and POMC mRNA was evaluated by semiquantitative in situ hybridization analysis. We observed a significant diurnal rhythm in AGRP mRNA expression, with a marked peak at 2200 h (4 h after lights off) and a trough at 1000 h (4 h after lights on), consistent with the overall day-night rhythm of food intake. In contrast, POMC mRNA levels did not show a significant fluctuation across the diurnal cycle, although there was a tendency for levels to decrease after the onset of the dark cycle. Corticosterone secretion temporally coincided with the rising phase of AGRP mRNA expression. Depletion of corticosterone by adrenalectomy abolished the AGRP diurnal rhythm by suppressing the nighttime expression, but did not alter the feeding rhythm. Exposure of adrenalectomized rats to constant corticosterone replacement (10 or 50 mg continuous release corticosterone pellet) resulted in fixed AGRP mRNA expression throughout the 12-h light, 12-h dark cycle. A relatively high level of corticosterone (50 mg) significantly increased AGRP mRNA expression, with a positive correlation between these two measures. These results indicate that 1) the diurnal expression of AGRP mRNA is regulated by corticosterone independently of the light/dark cue; and 2) a normal endogenous corticosterone rhythm is required for generating the diurnal AGRP rhythm.

  13. A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging.

    PubMed

    Jiang, Han; Moore, Sarah J; Liu, Shuanglong; Liu, Hongguang; Miao, Zheng; Cochran, Frank V; Liu, Yang; Tian, Mei; Cochran, Jennifer R; Zhang, Hong; Cheng, Zhen

    2013-02-01

    A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the α(v)β(3) integrin receptor with high affinity and specificity. In the current study, an (18)F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2-(18/19)F-fluoropropionate ((18/19)F-NFP) to produce the fluorinated peptide, (18/19)F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and (19)F-FP-AgRP-7C to human glioblastoma U87MG cells that overexpress α(v)β(3) integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with (18)F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and (19)F-FP-AgRP-7C specifically competed with (125)I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC(50)) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that (18)F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of (18)F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. (18)F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios

  14. Deletion of agouti-related protein blunts ethanol self-administration and binge-like drinking in mice.

    PubMed

    Navarro, M; Cubero, I; Ko, L; Thiele, T E

    2009-06-01

    The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Recent pharmacological and genetic evidence suggests that melanocortin receptor (MCR) signaling modulates neurobiological responses to ethanol and ethanol intake. Agouti-related protein (AgRP) is synthesized by neurons in the arcuate nucleus of the hypothalamus and is a natural antagonist of MCRs. Because central administration of the functionally active AgRP fragment AgRP-(83-132) increases ethanol intake by C57BL/6 J mice, we determined if mutant mice lacking normal production of AgRP (AgRP(-/-)) and maintained on a C57BL/6 J genetic background would show reduced self-administration of ethanol relative to littermate wild-type (AgRP(+/+)) mice. AgRP(-/-) mice showed reduced 8% (v/v) ethanol-reinforced lever-pressing behavior relative to AgRP(+/+) mice in daily 2-h sessions, but normal sucrose-, saccharin- and water-reinforced lever-pressing. Similarly, AgRP(-/-) mice showed reduced consumption of 8% ethanol in a two-bottle limited access test (2 h/day), although this effect was largely sex-dependent. Using drinking-in-the-dark (DID) procedures, AgRP(-/-) mice showed blunted binge-like drinking of 20% (v/v) ethanol which was associated with lower blood ethanol levels (85 mg/dl) relative to AgRP(+/+) mice (133 mg/dl) after 4 h of intake. AgRP(-/-) mice showed normal ethanol metabolism and did not show altered sensitivity to the sedative effects of ethanol. These observations with genetically altered mice are consistent with previous pharmacological data and suggest that endogenous AgRP signaling modulates the reinforcing properties of ethanol and binge-like ethanol drinking.

  15. Proopiomelanocortin, agouti-related protein, and leptin in human cerebrospinal fluid: correlations with body weight and adiposity.

    PubMed

    Page-Wilson, Gabrielle; Meece, Kana; White, Anne; Rosenbaum, Michael; Leibel, Rudolph L; Smiley, Richard; Wardlaw, Sharon L

    2015-09-01

    Leptin and its neuronal targets, which produce proopiomelanocortin (POMC) and agouti-related protein (AgRP), regulate energy balance. This study characterized leptin, POMC, and AgRP in the cerebrospinal fluid (CSF) of 47 healthy human subjects, 23 lean and 24 overweight/obese (OW/OB), as related to BMI, adiposity, plasma leptin, soluble leptin receptor (s-OB-R), and insulin. POMC was measured since the POMC prohormone is the predominant POMC peptide in CSF and correlates with hypothalamic POMC in rodents. Plasma AgRP was similarly characterized. CSF leptin was 83-fold lower than in plasma and correlated strongly with BMI, body fat, and insulin. The relative amount of leptin transported into CSF declined with increasing BMI, ranging from 4.5 to 0.52%, consistent with a saturable transport mechanism. CSF sOB-R was 78-fold lower than in plasma and correlated negatively with plasma and CSF leptin. CSF POMC was higher in lean vs. OW/OB subjects (P < 0.001) and correlated negatively with CSF leptin (r = -0.60, P < 0.001) and with plasma leptin, insulin, BMI, and adiposity. CSF AgRP was not different in lean vs. OW/OB; however, plasma AgRP was higher in lean subjects (P = 0.001) and correlated negatively with BMI, adiposity, leptin, insulin, and HOMA (P < 0.005). Thus, CSF measurements may provide useful biomarkers for brain leptin and POMC activity. The striking negative correlation between CSF leptin and POMC could be secondary to leptin resistance and/or neuronal changes associated with obesity but may also indicate that POMC plays a primary role in regulating body weight and adiposity. The role of plasma AgRP as a neuroendocrine biomarker deserves further study.

  16. Phosphodiesterase inhibitor-dependent inverse agonism of agouti-related protein on melanocortin 4 receptor in sea bass (Dicentrarchus labrax)

    PubMed Central

    Sánchez, Elisa; Rubio, Vera Cruz; Thompson, Darren; Metz, Juriaan; Flik, Gert; Millhauser, Glenn L.; Cerdá-Reverter, José Miguel

    2009-01-01

    The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor mainly expressed in the central nervous system of vertebrates. Activation of the MC4R leads to a decrease in food intake, whereas inactivating mutations are a genetic cause of obesity. The binding of agouti-related protein (AGRP) reduces not only agonist-stimulated cAMP production (competitive antagonist) but also the basal activity of the receptor, as an inverse agonist. Transgenic zebrafish overexpressing AGRP display increased food intake and linear growth, indicative of a physiological role for the melanocortin system in the control of the energy balance in fish. We report on the cloning, pharmacological characterization, tissue distribution, and detailed brain mapping of a sea bass (Dicentrarchus labrax) MC4R ortholog. Sea bass MC4R is profusely expressed within food intake-controlling pathways of the fish brain. However, the activity of the melanocortin system during progressive fasting does not depend on the hypothalamic/pituitary proopiomelanocortin (POMC) and MC4R expression, which suggests that sea bass MC4R is constitutively activated and regulated by AGRP binding. We demonstrate that AGRP acts as competitive antagonist and reduces MTII-induced cAMP production. AGRP also decreases the basal activity of the receptor as an inverse agonist. This observation suggests that MC4R is constitutively active and supports the evolutionary conservation of the AGRP/MC4R interactions. The inverse agonism, but not the competitive antagonism, depends on the presence of a phosphodiesterase inhibitor (IBMX). This suggests that inverse agonism and competitive antagonism operate through different intracellular signaling pathways, a view that opens up new targets for the treatment of melanocortin-induced metabolic syndrome. PMID:19225141

  17. A polymorphism in the human agouti-related protein is associated with late-onset obesity.

    PubMed

    Argyropoulos, George; Rankinen, Tuomo; Neufeld, Doni R; Rice, Treva; Province, Michael A; Leon, Arthur S; Skinner, James S; Wilmore, Jack H; Rao, D C; Bouchard, Claude

    2002-09-01

    The mouse agouti-related protein (AGRP) is a powerful appetite effector that results in hyperphagia and the development of obesity when administered intracerebroventricularly or when overexpressed in transgenic mice. Animal studies have also shown that exogenous administration of AGRP predisposes toward hedonic intake of high fat and high sucrose diets. The human ortholog (hAGRP) maps on chromosome 16q22 and has similar physiological properties, as tested in animal models. A polymorphism was identified in the third exon of hAGRP, c.199G-->A, that resulted in a nonconservative amino acid substitution, Ala(67)Thr. Computational analysis of the protein showed significant differences in the coils of the two polymorphic isoforms of the protein. Human studies showed no genotype effects in individuals with a mean age of 25 yr. However, the G/G genotype was significantly associated with fatness and abdominal adiposity in the parental population with a mean age of 53 yr. The c.199G-->A polymorphism in hAGRP could, therefore, play a role in the development of human obesity in an age-dependent fashion.

  18. Agouti-related protein increases food hoarding more than food intake in Siberian hamsters.

    PubMed

    Day, Diane E; Bartness, Timothy J

    2004-01-01

    Agouti-related protein (AgRP), an endogenous melanocortin 3/4 receptor antagonist, appears to play an important role in the control of food intake and energy balance because exogenous administration in rats and overexpression in mice result in hyperphagia and body mass gain. Furthermore, arcuate nucleus AgRP mRNA is increased with fasting in laboratory rats and mice and is decreased with refeeding. In Siberian hamsters, fasting also increases arcuate nucleus AgRP mRNA, but these animals increase food hoarding, rather than food intake with refeeding. Therefore, we tested whether exogenous AgRP increased food hoarding in this species. Hamsters were trained in a hoarding/foraging apparatus to run a programmed number of wheel revolutions to earn food pellets. Four doses of AgRP-(83-132) or vehicle were injected into the third ventricle at the beginning of the dark phase, and food hoarding, food intake, and foraging were measured at various time points subsequently. Overall, food hoarding was stimulated as much as 10 times more than food intake, and both responses occurred as early as 1 h after injection. Food hoarding was increased the greatest at the lowest dose (0.1 nmol), whereas food intake was increased the greatest at the second lowest dose (1 nmol). Food intake and especially food hoarding were increased up to seven days after the AgRP injections. Foraging was increased at all AgRP doses except the highest dose (100 nmol). These results suggest that AgRP triggers the search for food in this species, and once they find it, hoarding predominates over eating.

  19. Interleukin 6 Deficiency Modulates the Hypothalamic Expression of Energy Balance Regulating Peptides during Pregnancy in Mice

    PubMed Central

    Pazos, Patricia; Lima, Luis; Casanueva, Felipe F.; Diéguez, Carlos; García, María C.

    2013-01-01

    Pregnancy is associated with hyperphagia, increased adiposity and multiple neuroendocrine adaptations. Maternal adipose tissue secretes rising amounts of interleukin 6 (IL6), which acts peripherally modulating metabolic function and centrally increasing energy expenditure and reducing body fat. To explore the role of IL6 in the central mechanisms governing dam's energy homeostasis, early, mid and late pregnant (gestational days 7, 13 and 18) wild-type (WT) and Il6 knockout mice (Il6-KO) were compared with virgin controls at diestrus. Food intake, body weight and composition as well as indirect calorimetry measurements were performed in vivo. Anabolic and orexigenic peptides: neuropeptide Y (Npy) and agouti-related peptide (Agrp); and catabolic and anorectic neuropeptides: proopiomelanocortin (Pomc), corticotrophin and thyrotropin-releasing hormone (Crh and Trh) mRNA levels were determined by in situ hybridization. Real time-PCR and western-blot were used for additional tissue gene expression and protein studies. Non-pregnant Il6-KO mice were leaner than WT mice due to a decrease in fat but not in lean body mass. Pregnant Il6-KO mice had higher fat accretion despite similar body weight gain than WT controls. A decreased fat utilization in absence of Il6 might explain this effect, as shown by increased respiratory exchange ratio (RER) in virgin Il6-KO mice. Il6 mRNA levels were markedly enhanced in adipose tissue but reduced in hypothalamus of mid and late pregnant WT mice. Trh expression was also stimulated at gestational day 13 and lack of Il6 blunted this effect. Conversely, in late pregnant mice lessened hypothalamic Il6 receptor alpha (Il6ra), Pomc and Crh mRNA were observed. Il6 deficiency during this stage up-regulated Npy and Agrp expression, while restoring Pomc mRNA levels to virgin values. Together these results demonstrate that IL6/IL6Ra system modulates Npy/Agrp, Pomc and Trh expression during mouse pregnancy, supporting a role of IL6 in the central

  20. Peroxisome proliferator-activated receptor γ controls ingestive behavior, agouti-related protein, and neuropeptide Y mRNA in the arcuate hypothalamus.

    PubMed

    Garretson, John T; Teubner, Brett J W; Grove, Kevin L; Vazdarjanova, Almira; Ryu, Vitaly; Bartness, Timothy J

    2015-03-18

    Peroxisome proliferator-activated receptor γ (PPARγ) is clinically targeted for type II diabetes treatment; however, rosiglitazone (ROSI), a PPARγ agonist, increases food intake and body/fat mass as side-effects. Mechanisms for these effects and the role of PPARγ in feeding are not understood. Therefore, we tested this role in Siberian hamsters, a model of human energy balance, and C57BL/6 mice. We tested the following: (1) how ROSI and/or GW9662 (2-chloro-5-nitro-N-phenylbenzamide; PPARγ antagonist) injected intraperitoneally or into the third ventricle (3V) affected Siberian hamster feeding behaviors; (2) whether food deprivation (FD) co-increases agouti-related protein (AgRP) and PPARγ mRNA expression in Siberian hamsters and mice; (3) whether intraperitoneally administered ROSI increases AgRP and NPY in ad libitum-fed animals; (4) whether intraperitoneally administered PPARγ antagonism blocks FD-induced increases in AgRP and NPY; and finally, (5) whether intraperitoneally administered PPARγ modulation affects plasma ghrelin. Third ventricular and intraperitoneally administered ROSI increased food hoarding and intake for 7 d, an effect attenuated by 3V GW9662, and also prevented (intraperitoneal) FD-induced feeding. FD hamsters and mice increased AgRP within the arcuate hypothalamic nucleus with concomitant increases in PPARγ exclusively within AgRP/NPY neurons. ROSI increased AgRP and NPY similarly to FD, and GW9662 prevented FD-induced increases in AgRP and NPY in both species. Neither ROSI nor GW9662 affected plasma ghrelin. Thus, we demonstrated that PPARγ activation is sufficient to trigger food hoarding/intake, increase AgRP/NPY, and possibly is necessary for FD-induced increases in feeding and AgRP/NPY. These findings provide initial evidence that FD-induced increases in AgRP/NPY may be a direct PPARγ-dependent process that controls ingestive behaviors.

  1. Peroxisome proliferator-activated receptor γ controls ingestive behavior, agouti-related protein, and neuropeptide Y mRNA in the arcuate hypothalamus.

    PubMed

    Garretson, John T; Teubner, Brett J W; Grove, Kevin L; Vazdarjanova, Almira; Ryu, Vitaly; Bartness, Timothy J

    2015-03-18

    Peroxisome proliferator-activated receptor γ (PPARγ) is clinically targeted for type II diabetes treatment; however, rosiglitazone (ROSI), a PPARγ agonist, increases food intake and body/fat mass as side-effects. Mechanisms for these effects and the role of PPARγ in feeding are not understood. Therefore, we tested this role in Siberian hamsters, a model of human energy balance, and C57BL/6 mice. We tested the following: (1) how ROSI and/or GW9662 (2-chloro-5-nitro-N-phenylbenzamide; PPARγ antagonist) injected intraperitoneally or into the third ventricle (3V) affected Siberian hamster feeding behaviors; (2) whether food deprivation (FD) co-increases agouti-related protein (AgRP) and PPARγ mRNA expression in Siberian hamsters and mice; (3) whether intraperitoneally administered ROSI increases AgRP and NPY in ad libitum-fed animals; (4) whether intraperitoneally administered PPARγ antagonism blocks FD-induced increases in AgRP and NPY; and finally, (5) whether intraperitoneally administered PPARγ modulation affects plasma ghrelin. Third ventricular and intraperitoneally administered ROSI increased food hoarding and intake for 7 d, an effect attenuated by 3V GW9662, and also prevented (intraperitoneal) FD-induced feeding. FD hamsters and mice increased AgRP within the arcuate hypothalamic nucleus with concomitant increases in PPARγ exclusively within AgRP/NPY neurons. ROSI increased AgRP and NPY similarly to FD, and GW9662 prevented FD-induced increases in AgRP and NPY in both species. Neither ROSI nor GW9662 affected plasma ghrelin. Thus, we demonstrated that PPARγ activation is sufficient to trigger food hoarding/intake, increase AgRP/NPY, and possibly is necessary for FD-induced increases in feeding and AgRP/NPY. These findings provide initial evidence that FD-induced increases in AgRP/NPY may be a direct PPARγ-dependent process that controls ingestive behaviors. PMID:25788674

  2. Peroxisome Proliferator-Activated Receptor γ Controls Ingestive Behavior, Agouti-Related Protein, and Neuropeptide Y mRNA in the Arcuate Hypothalamus

    PubMed Central

    Garretson, John T.; Teubner, Brett J.W.; Grove, Kevin L.; Vazdarjanova, Almira; Ryu, Vitaly

    2015-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) is clinically targeted for type II diabetes treatment; however, rosiglitazone (ROSI), a PPARγ agonist, increases food intake and body/fat mass as side-effects. Mechanisms for these effects and the role of PPARγ in feeding are not understood. Therefore, we tested this role in Siberian hamsters, a model of human energy balance, and C57BL/6 mice. We tested the following: (1) how ROSI and/or GW9662 (2-chloro-5-nitro-N-phenylbenzamide; PPARγ antagonist) injected intraperitoneally or into the third ventricle (3V) affected Siberian hamster feeding behaviors; (2) whether food deprivation (FD) co-increases agouti-related protein (AgRP) and PPARγ mRNA expression in Siberian hamsters and mice; (3) whether intraperitoneally administered ROSI increases AgRP and NPY in ad libitum-fed animals; (4) whether intraperitoneally administered PPARγ antagonism blocks FD-induced increases in AgRP and NPY; and finally, (5) whether intraperitoneally administered PPARγ modulation affects plasma ghrelin. Third ventricular and intraperitoneally administered ROSI increased food hoarding and intake for 7 d, an effect attenuated by 3V GW9662, and also prevented (intraperitoneal) FD-induced feeding. FD hamsters and mice increased AgRP within the arcuate hypothalamic nucleus with concomitant increases in PPARγ exclusively within AgRP/NPY neurons. ROSI increased AgRP and NPY similarly to FD, and GW9662 prevented FD-induced increases in AgRP and NPY in both species. Neither ROSI nor GW9662 affected plasma ghrelin. Thus, we demonstrated that PPARγ activation is sufficient to trigger food hoarding/intake, increase AgRP/NPY, and possibly is necessary for FD-induced increases in feeding and AgRP/NPY. These findings provide initial evidence that FD-induced increases in AgRP/NPY may be a direct PPARγ-dependent process that controls ingestive behaviors. PMID:25788674

  3. Bacterial expression of self-assembling peptide hydrogelators

    NASA Astrophysics Data System (ADS)

    Sonmez, Cem

    For tissue regeneration and drug delivery applications, various architectures are explored to serve as biomaterial tools. Via de novo design, functional peptide hydrogel materials have been developed as scaffolds for biomedical applications. The objective of this study is to investigate bacterial expression as an alternative method to chemical synthesis for the recombinant production of self-assembling peptides that can form rigid hydrogels under physiological conditions. The Schneider and Pochan Labs have designed and characterized a 20 amino acid beta-hairpin forming amphiphilic peptide containing a D-residue in its turn region (MAX1). As a result, this peptide must be prepared chemically. Peptide engineering, using the sequence of MAX1 as a template, afforded a small family of peptides for expression (EX peptides) that have different turn sequences consisting of natural amino acids and amenable to bacterial expression. Each sequence was initially chemically synthesized to quickly assess the material properties of its corresponding gel. One model peptide EX1, was chosen to start the bacterial expression studies. DNA constructs facilitating the expression of EX1 were designed in such that the peptide could be expressed with different fusion partners and subsequently cleaved by enzymatic or chemical means to afford the free peptide. Optimization studies were performed to increase the yield of pure peptide that ultimately allowed 50 mg of pure peptide to be harvested from one liter of culture, providing an alternate means to produce this hydrogel-forming peptide. Recombinant production of other self-assembling hairpins with different turn sequences was also successful using this optimized protocol. The studies demonstrate that new beta-hairpin self-assembling peptides that are amenable to bacterial production and form rigid hydrogels at physiological conditions can be designed and produced by fermentation in good yield at significantly reduced cost when compared to

  4. Expression of peptide YY by human blood leukocytes.

    PubMed

    Holler, Julia Pia Natascha; Schmitz, Jessica; Roehrig, Rainer; Wilker, Sigrid; Hecker, Andreas; Padberg, Winfried; Grau, Veronika

    2014-08-01

    Peptide YY is produced by L cells in the mucosa of the distal ileum, colon, and rectum and may have systemic and paracrine functions. We hypothesized that peptide YY is expressed by peripheral blood mononuclear cells. The purpose of the present study was to evaluate the expression of peptide YY mRNA and peptide by peripheral blood mononuclear cells and differentiated THP-1 cells after lipopolysaccharide treatment as an in vitro model of inflammation. Blood was drawn by venipuncture from 18- to 63-year-old healthy male blood donors (n=63); peptide YY mRNA expression levels were detected in peripheral blood mononuclear cells from all healthy male subjects. In 3 subjects, peripheral blood mononuclear cells were cultured for 3 and 24h and peptide YY was detected in the cell culture supernatant. In human monocytic THP-1 cells treated with phorbol-12-myristate-13-acetate to induce differentiation to macrophages, treatment with lipopolysaccharide caused down-regulation of peptide YY mRNA levels. In summary, freshly isolated peripheral blood mononuclear cells from healthy humans expressed peptide YY. In vitro data suggested that peptide YY expression is down-regulated by differentiation of monocytes to macrophages and proinflammatory stimuli.

  5. Surface expression, peptide repertoire, and thermostability of chicken class I molecules correlate with peptide transporter specificity

    PubMed Central

    Tregaskes, Clive A.; Harrison, Michael; Sowa, Anna K.; van Hateren, Andy; Hunt, Lawrence G.; Vainio, Olli; Kaufman, Jim

    2016-01-01

    The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek’s disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC). PMID:26699458

  6. Plant biofarming: novel insights for peptide expression in heterologous systems.

    PubMed

    Viana, Antônio Américo Barbosa; Pelegrini, Patrícia Barbosa; Grossi-de-Sá, Maria Fátima

    2012-01-01

    Peptide expression methods have been widely studied and developed from many different biological sources. The cultivation ofprokaryotic and eukaryotic cells has proven to be efficient for the expression of foreign peptides in several heterologous systems, including bacteria, insects, yeasts, and mammals. Earlier reports brought up new insights for the improvement of expressed products to not only increase the production rate of desired peptides but also reproduce desirable post-translational modifications and even to reduce the risk of allergenicity when those products are aimed for human use. The development of bioreactor systems provided the optimization of cell growth conditions to scale up the amounts of expressed peptides. On the other hand, different cell systems and mutants provided a plethora of possible peptide modifications. Hence, in this report, we describe the many organisms and systems used for the large scale production of several macromolecules with relevance in health and agriculture. We also bring into discussion plant biofarming in the moss Physcomitrella patens and its recent adaptations, as a cost-effective and efficient approach in the production of more complex heterologous proteins, given the fact that its glycosylation pattern can be engineered to avoid allergenicity to humans (common to plant-derived glycoproteins). PMID:23193604

  7. Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

    PubMed

    Hahm, S; Mizuno, T M; Wu, T J; Wisor, J P; Priest, C A; Kozak, C A; Boozer, C N; Peng, B; McEvoy, R C; Good, P; Kelley, K A; Takahashi, J S; Pintar, J E; Roberts, J L; Mobbs, C V; Salton, S R

    1999-07-01

    To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

  8. Expression of the mammalian peptide hormone obestatin in Trichoderma reesei.

    PubMed

    Sun, Angela; Peterson, Robyn; Te'o, Junior; Nevalainen, Helena

    2016-01-25

    The filamentous fungus Trichoderma reesei is an expression host widely exploited for the production of recombinant proteins. However, its capacity for expressing small peptides (<20 kDa) has remained largely uncharted to date. In this work, we have produced the hormone peptide obestatin fused to the hydrophobin I tag (Obe-HFBI), using the T. reesei cellobiohydrolase I core (CBHI) or xylanase 2 (XYN2) pro-region as a carrier and the cbh1 promoter for gene expression, in high protein-low protease producing mutant strains T. reesei Rut-C30 and HEPI. The yield of obestatin was improved from about 300 ng/ml to up to 5.5 μg/ml through adaptive laboratory evolution and modifications to the cultivation strategy, which included adjustments of the type and ratio of carbon and nitrogen sources used in the medium. The successful expression of Obe-HFBI demonstrated the potential of T. reesei as an expression host for small peptides and further enhancement of the recombinant yield through modification of culture conditions. PMID:26341165

  9. Expression of tracheal antimicrobial peptide in bovine mammary epithelial cells.

    PubMed

    López-Meza, Joel E; Gutiérrez-Barroso, Angelina; Ochoa-Zarzosa, Alejandra

    2009-08-01

    The production of antimicrobial peptides is an important key of innate immunity. Tracheal antimicrobial peptide (TAP) expression has been reported in bovine tracheal epithelial cells and it can be modulated by bacterial infection or bacterial components. In mammary gland TAP expression has been reported, but the cell type that produces it is unknown. The objective of this work was to evaluate if bovine mammary epithelial cells (bMEC) express TAP mRNA, and evaluate the regulation of its expression in response to Staphylococcus aureus infection, bovine prolactin (bPRL) or acetyl salicylic acid (ASA). By retrotranscription and PCR, we demonstrated that bMEC express TAP mRNA. bMEC infected with live S. aureus down-regulates TAP expression, whereas the challenge with gentamicin-killed S. aureus up-regulates it. Also, bPRL do not significantly modify TAP expression, but in the presence of 5 mM ASA it was down-regulated, suggesting that nuclear factor kappaB (NF-kappaB) pathway can be involved in its regulation. PMID:19181355

  10. Hypothalamic neuropeptide gene expression during recovery from food restriction superimposed on short-day photoperiod-induced weight loss in the Siberian hamster.

    PubMed

    Archer, Zoë A; Moar, Kim M; Logie, Tracy J; Reilly, Laura; Stevens, Valerie; Morgan, Peter J; Mercer, Julian G

    2007-09-01

    Previously, 40% food restriction of male Siberian hamsters over 21 days in short-day (SD) photoperiod induced characteristic changes in expression of hypothalamic arcuate nucleus energy balance genes; mRNAs for neuropeptide Y, agouti-related peptide, and leptin receptor were upregulated, and those of proopiomelanocortin and cocaine- and amphetamine-regulated transcript were depressed. The present study examined the effect of refeeding hamsters for 6 days (approximately 50% recovery of weight differential) or 19 days (resumption of appropriate weight trajectory). Hyperphagia continued throughout refeeding, but differences in fat pad weights and leptin levels had disappeared after 19 days. Cocaine- and amphetamine-regulated transcript gene expression was depressed by prior restriction in both refed groups. The depressive effect of prior restriction on proopiomelanocortin gene expression had disappeared after 19 days of refeeding. There was no effect of prior food restriction on neuropeptide Y or agouti-related peptide gene expression. Expression of the anorexigenic brain-derived neurotrophic factor was downregulated in the ventromedial nucleus after SD exposure for 12 wk. In the SD food restriction study, there were effects of photoperiod on brain-derived neurotrophic factor gene expression but not of prior food restriction. Hypothalamic energy balance genes in the hamster respond asynchronously to return to a seasonally appropriate body weight. The achievement of this weight rather than the weight at which caloric restriction was imposed is the critical factor. The differential responses of hypothalamic energy balance genes to food restriction and refeeding are poorly characterized in any species, a critical issue given their potential relevance to human weight loss strategies that involve caloric restriction.

  11. Differential gene expression of the three natriuretic peptides and natriuretic peptide receptor subtypes in human liver.

    PubMed Central

    Vollmar, A M; Paumgartner, G; Gerbes, A L

    1997-01-01

    BACKGROUND: Various effects of atrial natriuretic peptide (ANP) on the liver have been observed. However, there is limited information about the types of receptors for natriuretic peptides expressed by the human liver. AIM: To investigate gene expression of the three NP receptor types (NPR) as well as of the NP in human liver. METHODS: Presence of mRNA coding for all three NPR and for ANP, brain and C-type natriuretic peptide (BNP, CNP) was investigated by reverse transcription-polymerase chain reaction (RT-PCR). Human liver tissues and hepatocellular carcinoma tissues were examined. RESULTS: Specific PCR products for all three NPR, namely NPR-A, B, and C, could be detected. Moreover, ANP and CNP, but not BNP mRNA was detectable. The concentration of ANP transcripts was up to fivefold higher in hepatocellular carcinoma compared with non-tumorous liver tissue of the same subjects. No difference in the expression of NP receptors relative to GAPDH mRNA of tumorous and non-tumorous tissue was observed except of slightly increased NPR-A transcripts. CONCLUSION: These data show that NPR transcripts are coexpressed with ANP and CNP mRNA in the human liver. This provides evidence for a local NP system in the human liver. Images PMID:9155593

  12. Immune Signaling and Antimicrobial Peptide Expression in Lepidoptera.

    PubMed

    Casanova-Torres, Ángel M; Goodrich-Blair, Heidi

    2013-09-01

    Many lepidopteran insects are agricultural pests that affect stored grains, food and fiber crops. These insects have negative ecological and economic impacts since they lower crop yield, and pesticides are expensive and can have off-target effects on beneficial arthropods. A better understanding of lepidopteran immunity will aid in identifying new targets for the development of specific insect pest management compounds. A fundamental aspect of immunity, and therefore a logical target for control, is the induction of antimicrobial peptide (AMP) expression. These peptides insert into and disrupt microbial membranes, thereby promoting pathogen clearance and insect survival. Pathways leading to AMP expression have been extensively studied in the dipteran Drosophila melanogaster. However, Diptera are an important group of pollinators and pest management strategies that target their immune systems is not recommended. Recent advances have facilitated investigation of lepidopteran immunity, revealing both conserved and derived characteristics. Although the general pathways leading to AMP expression are conserved, specific components of these pathways, such as recognition proteins have diverged. In this review we highlight how such comparative immunology could aid in developing pest management strategies that are specific to agricultural insect pests.

  13. Immune Signaling and Antimicrobial Peptide Expression in Lepidoptera

    PubMed Central

    Casanova-Torres, Ángel M.; Goodrich-Blair, Heidi

    2013-01-01

    Many lepidopteran insects are agricultural pests that affect stored grains, food and fiber crops. These insects have negative ecological and economic impacts since they lower crop yield, and pesticides are expensive and can have off-target effects on beneficial arthropods. A better understanding of lepidopteran immunity will aid in identifying new targets for the development of specific insect pest management compounds. A fundamental aspect of immunity, and therefore a logical target for control, is the induction of antimicrobial peptide (AMP) expression. These peptides insert into and disrupt microbial membranes, thereby promoting pathogen clearance and insect survival. Pathways leading to AMP expression have been extensively studied in the dipteran Drosophila melanogaster. However, Diptera are an important group of pollinators and pest management strategies that target their immune systems is not recommended. Recent advances have facilitated investigation of lepidopteran immunity, revealing both conserved and derived characteristics. Although the general pathways leading to AMP expression are conserved, specific components of these pathways, such as recognition proteins have diverged. In this review we highlight how such comparative immunology could aid in developing pest management strategies that are specific to agricultural insect pests. PMID:25861461

  14. Heterologous expression of five disulfide-bonded insecticidal spider peptides.

    PubMed

    Estrada, Georgina; Silva, Anita O; Villegas, Elba; Ortiz, Ernesto; Beirão, Paulo S L; Corzo, Gerardo

    2016-09-01

    The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana. PMID:27263806

  15. Heterologous expression of five disulfide-bonded insecticidal spider peptides.

    PubMed

    Estrada, Georgina; Silva, Anita O; Villegas, Elba; Ortiz, Ernesto; Beirão, Paulo S L; Corzo, Gerardo

    2016-09-01

    The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana.

  16. Suppression of Antimicrobial Peptide Expression by Ureaplasma Species

    PubMed Central

    Crabb, Donna M.; Dai, Yuling; Chen, Yuying; Waites, Ken B.; Atkinson, T. Prescott

    2014-01-01

    Ureaplasma species commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome of Ureaplasma infections. THP-1 cells, a human monocytoid tumor line, were cocultured with Ureaplasma parvum and U. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR. In vitro antimicrobial activities of synthetic AMPs against Ureaplasma spp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation with U. parvum and U. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1, DEFA5, DEFA6, and CAMP), was significantly downregulated compared to that of TNFA and IL-8, which were upregulated. In vitro flow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity against Ureaplasma spp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation with U. parvum infection. No DNA methylation status changes were detected upon Ureaplasma infection. In conclusion, AMPs have in vitro activity against Ureaplasma spp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization. PMID:24491573

  17. Chamber-dependent circadian expression of cardiac natriuretic peptides.

    PubMed

    Goetze, Jens Peter; Georg, Birgitte; Jørgensen, Henrik L; Fahrenkrug, Jan

    2010-02-25

    Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) have important local functions within the myocardium, where they protect against accelerated fibrosis. As circadian expression of cardiac natriuretic peptides could be of importance in local cardiac protection against disease, we examined the diurnal changes of the mRNAs encoding ANP, BNP, and their common receptor NPR-A in atrial and ventricular myocardium. Forty eight mice were killed at the following ZT times: 4, 8, 12, 16, 20, and 24, where ZT designates Zeitgeber; ZT 0 corresponds to lights ON and ZT 12 corresponds to lights OFF. Eight animals (4 males and 4 females) were included at each time point. Another 48 animals were killed during the second cycle of dark/dark (designated Circadian Time or CT: CT 4, CT 8, CT 12, CT 16, CT 20, and CT 24). The cellular contents of the clock genes Per1 and Bmal1 as well as ANP, BNP, and their common receptor (NPR-A) were determined using RT-PCR. Per1 and Bmal1 mRNA contents oscillated in antiphase in both atrial and ventricular regions, where Bmal1 mRNA peaked 12h out of phase relative to Per1 mRNA. ANP and NPR-A atrial mRNA contents revealed borderline significant diurnal changes, whereas ventricular BNP mRNA contents exhibited pronounced oscillation during constant darkness with nadir at CT 12 (P<0.0001). In conclusion, we report a chamber-dependent circadian profile of cardiac BNP mRNA contents, which is not paralleled by the related ANP gene. Our findings suggest that the BNP mRNA pattern could be associated with increased cardiac susceptibility and response to disease.

  18. [Expression and adjuvant effects of the fusion peptide TBP5].

    PubMed

    Wang, Chen; Guo, Xiangling; Li, Xiaokang; Wu, Tingcai; Li, Deyuan; Chen, Puyan

    2015-05-01

    Thymopentin (TP5) and bursopentin (BP5) are both immunopotentiators. To explore whether the TP5-BP5 fusion peptide (TBP5) has adjuvant activity or not, we cloned the TBP5 gene and confirmed that the TBP5 gene in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. TBP5 significantly promoted the proliferation of thymic and splenic lymphocytes of mice. The potential adjuvant activity of the TBP5 was examined in mice by coinjecting TBP5 and H9N2 avian influenza virus (AIV) inactivated vaccine. HI antibody titers, HA antibodies and cytokines levels (IL-4 and IFN-γ) were determined. We found that TBP5 markedly elevated serum HI titers and HA antibody levels, induced the secretion of both IL-4 and IFN-γ cytokines. Furthermore, virus challenge experiments confirmed that TBP5 contributed to inhibition replication of the virus [H9N2 AIV (A/chicken/Jiangsu/NJ07/05)] from mouse lungs. Altogether, these findings suggest that TBP5 may be an effective adjuvant for avian vaccine and that this study provides a reference for further research on new vaccine adjuvants. PMID:26571686

  19. Purification and characterization of a plant antimicrobial peptide expressed in Escherichia coli.

    PubMed

    Harrison, S J; McManus, A M; Marcus, J P; Goulter, K C; Green, J L; Nielsen, K J; Craik, D J; Maclean, D J; Manners, J M

    1999-03-01

    MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isolated from the nut kernel of Macadamia integrifolia. A DNA sequence encoding MiAMP1 with an additional ATG start codon was cloned into a modified pET vector under the control of the T7 RNA polymerase promoter. The pET vector was cotransformed together with the vector pSB161, which expresses a rare arginine tRNA. The peptide was readily isolated in high yield from the insoluble fraction of the Escherichia coli extract. The purified peptide was shown to have an identical molecular weight to the native peptide by mass spectroscopy indicating that the N-terminal methionine had been cleaved. Analysis by NMR spectroscopy indicated that the refolded recombinant peptide had a similar overall three-dimensional structure to that of the native peptide. The peptide inhibited the growth of phytopathogenic fungi in vitro in a similar manner to the native peptide. To our knowledge, MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed in E. coli. This will permit a detailed structure-function analysis of the peptide and studies of its mode of action on phytopathogens.

  20. Purification and characterization of a plant antimicrobial peptide expressed in Escherichia coli.

    PubMed

    Harrison, S J; McManus, A M; Marcus, J P; Goulter, K C; Green, J L; Nielsen, K J; Craik, D J; Maclean, D J; Manners, J M

    1999-03-01

    MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isolated from the nut kernel of Macadamia integrifolia. A DNA sequence encoding MiAMP1 with an additional ATG start codon was cloned into a modified pET vector under the control of the T7 RNA polymerase promoter. The pET vector was cotransformed together with the vector pSB161, which expresses a rare arginine tRNA. The peptide was readily isolated in high yield from the insoluble fraction of the Escherichia coli extract. The purified peptide was shown to have an identical molecular weight to the native peptide by mass spectroscopy indicating that the N-terminal methionine had been cleaved. Analysis by NMR spectroscopy indicated that the refolded recombinant peptide had a similar overall three-dimensional structure to that of the native peptide. The peptide inhibited the growth of phytopathogenic fungi in vitro in a similar manner to the native peptide. To our knowledge, MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed in E. coli. This will permit a detailed structure-function analysis of the peptide and studies of its mode of action on phytopathogens. PMID:10049672

  1. Increased food intake in growth hormone-transgenic common carp (Cyprinus carpio L.) may be mediated by upregulating Agouti-related protein (AgRP).

    PubMed

    Zhong, Chengrong; Song, Yanlong; Wang, Yaping; Zhang, Tanglin; Duan, Ming; Li, Yongming; Liao, Lanjie; Zhu, Zuoyan; Hu, Wei

    2013-10-01

    In fish, food intake and feeding behavior are crucial for survival, competition, growth and reproduction. Growth hormone (GH)-transgenic common carp exhibit an enhanced growth rate, increased food intake and higher feed conversion rate. However, the underlying molecular mechanisms of feeding regulation in GH-transgenic (TG) fish are not clear. In this study, we observed feeding behavior of TG and non-transgenic (NT) common carp, and analyzed the mRNA expression levels of NPY, AgRP I, orexin, POMC, CCK, and CART I in the hypothalamus and telencephalon after behavioral observation. We detected similar gene expression levels in the hypothalamus of TG and NT common carp, which had been cultured in the field at the same age. Furthermore, we tested the effects of GH on hypothalamus fragments in vitro to confirm our findings. We demonstrated that TG common carp displayed increased food intake and reduced food consumption time, which were associated with a marked increase in hypothalamic AgRP I mRNA expression. Our results suggest that elevated GH levels may influence food intake and feeding behavior by upregulating the hypothalamic orexigenic factor AgRP I in GH-transgenic common carp. PMID:23583469

  2. A Systematic Analysis of Drosophila Regulatory Peptide Expression in Enteroendocrine Cells

    PubMed Central

    Chen, Ji; Kim, Seol-min; Kwon, Jae Young

    2016-01-01

    The digestive system is gaining interest as a major regulator of various functions including immune defense, nutrient accumulation, and regulation of feeding behavior, aside from its conventional function as a digestive organ. The Drosophila midgut epithelium is completely renewed every 1–2 weeks due to differentiation of pluripotent intestinal stem cells in the midgut. Intestinal stem cells constantly divide and differentiate into enterocytes that secrete digestive enzymes and absorb nutrients, or enteroendocrine cells that secrete regulatory peptides. Regulatory peptides have important roles in development and metabolism, but study has mainly focused on expression and functions in the nervous system, and not much is known about the roles in endocrine functions of enteroendocrine cells. We systemically examined the expression of 45 regulatory peptide genes in the Drosophila midgut, and verified that at least 10 genes are expressed in the midgut enteroendocrine cells through RT-PCR, in situ hybridization, antisera, and 25 regulatory peptide-GAL transgenes. The Drosophila midgut is highly compartmentalized, and individual peptides in enteroendocrine cells were observed to express in specific regions of the midgut. We also confirmed that some peptides expressed in the same region of the midgut are expressed in mutually exclusive enteroendocrine cells. These results indicate that the midgut enteroendocrine cells are functionally differentiated into different subgroups. Through this study, we have established a basis to study regulatory peptide functions in enteroendocrine cells as well as the complex organization of enteroendocrine cells in the Drosophila midgut. PMID:27025390

  3. Design of Self-Assembling Peptide Hydrogelators Amenable to Bacterial Expression

    PubMed Central

    Sonmez, Cem; Nagy, Katelyn J.; Schneider, Joel P.

    2014-01-01

    Hydrogels formed from self-assembling peptides are finding use in tissue engineering and drug delivery applications. Given the notorious difficulties associated with producing self-assembling peptides by recombinant expression, most are typically prepared by chemical synthesis. Herein, we report the design of a family of self-assembling β-hairpin peptides amenable to efficient production using an optimized bacterial expression system. Expressing peptides, EX1, EX2 and EX3 contain identical eight-residue amphiphilic β-strands connected by varying turn sequences that are responsible for ensuring chain reversal and the proper intramolecular folding and consequent self-assembly of the peptide into a hydrogel network under physiological conditions. EX1 was initially used to establish and optimize the bacterial expression system by which all the peptides could be eventually individually expressed. Expression clones were designed to allow exploration of possible fusion partners and investigate both enzymatic and chemical cleavage as means to liberate the target peptide. A systematic analysis of possible expression systems followed by fermentation optimization lead to a system in which all three peptides could be expressed as fusions with BAD-BH3, the BH3 domain of the proapoptotic BAD (Bcl-2 Associated Death) Protein. CNBr cleavage followed by purification afforded 50, 31, and 15 mg/L yields of pure EX1, EX2 and EX3, respectively. CD spectroscopy, TEM, and rheological analysis indicate that these peptides fold and assembled into well-defined fibrils that constitute hydrogels having shear-thin/recovery properties. PMID:25453938

  4. Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using quantitative PCR technique, the relative transcriptional levels of seven channel catfish antimicrobial peptide (AMP) genes [NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability-increasing protein (BPI), cathepsin D, hepcidin, and liver-expressed antimicrobial peptide 2 ...

  5. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.

    PubMed

    Baeshen, Mohammed N; Bouback, Thamer A F; Alzubaidi, Mubarak A; Bora, Roop S; Alotaibi, Mohammed A T; Alabbas, Omar T O; Alshahrani, Sultan M; Aljohani, Ahmed A M; Munshi, Rayan A A; Al-Hejin, Ahmed; Ahmed, Mohamed M M; Redwan, Elrashdy M; Ramadan, Hassan A I; Saini, Kulvinder S; Baeshen, Nabih A

    2016-01-01

    Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system. PMID:27579308

  6. Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System

    PubMed Central

    Baeshen, Mohammed N.; Bouback, Thamer A. F.; Alzubaidi, Mubarak A.; Alabbas, Omar T. O.; Alshahrani, Sultan M.; Aljohani, Ahmed A. M.; Munshi, Rayan A. A.; Al-Hejin, Ahmed; Redwan, Elrashdy M.; Ramadan, Hassan A. I.; Saini, Kulvinder S.; Baeshen, Nabih A.

    2016-01-01

    Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system. PMID:27579308

  7. Expression and differential regulation of natriuretic peptides in mouse macrophages.

    PubMed Central

    Vollmar, A M; Schulz, R

    1995-01-01

    The coexpression of the natriuretic peptides ANP, BNP and CNP as well as their differential regulation in mouse macrophages was demonstrated by quantitative PCR, HPLC analysis, and specific radioimmunoassays. Exposure of peritoneal- and bone marrow-derived macrophages to various immunomodulators revealed that bacterial LPS strikingly increases (up to 300-fold) the mRNA coding for CNP as does zymosan (up to 15-fold). In this respect, neither the phorbol ester PMA nor the glucocorticoid dexamethasone had any effect. Examination of macrophages for ANP mRNA showed a similar response to LPS and zymosan, though only a three- to sixfold increase, confirming previous data. In contrast, the concentration of mRNA coding for brain natriuretic peptide in these cells was reduced by dexamethasone (up to twofold) as well as LPS (two- to fivefold). No change was observed upon challenge with zymosan or PMA. The findings at the mRNA level are complemented by their corresponding peptide products. Incubation of macrophages with LPS resulted in a two- and fivefold elevation of intracellular ANP and CNP immunoreactivity, respectively. The amount of peptides released from cells under these conditions was found increased for ANP (threefold) and CNP (10-fold). No changes were observed for both intra- and extracellular brain natriuretic peptide. The coexpression of natriuretic peptides in macrophages as well as their different regulations by immunomodulators suggest discrete functions of these peptides within the immune system. Images PMID:7769089

  8. Design and expression of a short peptide as an HIV detection probe

    SciTech Connect

    Lines, Jamie A.; Yu, Zhiqiang; Dedkova, Larisa M.; Chen, Shengxi

    2014-01-03

    Highlights: •We designed a short fusion peptide (FP-50) for in vivo expression. •This peptide is a very promising component for detection of gp120 protein. •The detectable level is about 20–200 times lower than previously published methods. •It is a novel probe to detect HIV-1 gp120 during early stages of HIV infection. -- Abstract: To explore a low-cost novel probe for HIV detection, we designed and prepared a 50-amino acid-length short fusion peptide (FP-50) via Escherichia coli in vivo expression. It was employed as a novel probe to detect HIV-1 gp120 protein. The detectable level of gp120 protein using the FP-50 peptide was approximately 20–200 times lower than previously published methods that used a pair of monoclonal antibodies. Thus, this short peptide is a very promising component for detection of gp120 protein during early stages of HIV infection.

  9. Expression systems for heterologous production of antimicrobial peptides.

    PubMed

    Parachin, Nádia Skorupa; Mulder, Kelly Cristina; Viana, Antônio Américo Barbosa; Dias, Simoni Campos; Franco, Octávio Luiz

    2012-12-01

    Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.

  10. Engineered Cystine-Knot Peptides That Bind αvβ3 Integrin With Antibody-Like Affinities

    PubMed Central

    Silverman, Adam P.; Levin, Aron M.; Lahti, Jennifer L.; Cochran, Jennifer R.

    2010-01-01

    The αvβ3 integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature, and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4 kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to αvβ3 integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a 6-amino acid loop of AgRP with a 9-amino acid loop containing the Arg-Gly-Asp (RGD) integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting (FACS) to identify clones with high affinity to detergent-solubilized αvβ3 integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing αvβ3 integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown bind specifically to αvβ3 integrins, and had only minimal or no binding to αvβ5, α5β1, and αiibβ3 integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally-occurring ligand for αvβ3 and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second generation libraries by individually randomizing these loops in one of the high affinity integrin-binding variants. Screening of these loop-randomized libraries against αvβ3 integrins resulted in peptides that retained high affinities for αvβ3 and had increased specificities for αvβ3 over αiibβ3 integrins. Collectively, these data

  11. Glucose-dependent insulinotropic peptide: structure of the precursor and tissue-specific expression in rat.

    PubMed Central

    Tseng, C C; Jarboe, L A; Landau, S B; Williams, E K; Wolfe, M M

    1993-01-01

    Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic beta cells in the presence of glucose. Approximately 7.8 x 10(5) recombinant clones of a neonatal rat intestinal cDNA library were screened by using plaque hybridization, and three clones were identified and sequenced with the dideoxynucleotide chain-termination method. The translated amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that rat GIP was derived by proteolytic processing of a 144-amino acid precursor polypeptide. The mature peptide is flanked by a 43-amino acid NH2-terminal peptide that contains a 21-amino acid signal peptide and by a 59-amino acid COOH-terminal peptide. Analysis of the nucleotide and amino acid sequence of rat GIP revealed only two substitutions from the known human GIP peptide. The use of high-stringency RNA blot-hybridization analysis of total RNA extracted from various organs demonstrated expression of the GIP gene in the duodenum and jejunum and, to a lesser extent, in the ileum. In addition, expression of the GIP gene was observed in the submandibular salivary gland both by RNA analysis and RIA. In response to duodenal perfusion of a 20% Lipomul meal for 60 min, duodenal mucosal GIP mRNA concentrations increased by 42.8% and 48.2% at 30 and 60 min, respectively. Images Fig. 1 Fig. 4 PMID:8446620

  12. Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression

    SciTech Connect

    Olszewski, Pawel K.; Fredriksson, Robert; Eriksson, Jenny D.; Mitra, Anaya; Radomska, Katarzyna J.; Gosnell, Blake A.; Solvang, Maria N.; Levine, Allen S.; Schioeth, Helgi B.

    2011-05-13

    Highlights: {yields} The majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto. {yields} The level of colocalization is similar in the male and female brain. {yields} Fto overexpression in hypothalamic neurons increases oxytocin mRNA levels by 50%. {yields} Oxytocin does not affect Fto expression through negative feedback mechanisms. -- Abstract: Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance through a functional relationship with specific genes encoding feeding-related peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin.

  13. Ovine trophoblasts express cathelicidin host defence peptide in response to infection.

    PubMed

    Coyle, Christopher; Wheelhouse, Nick; Jacques, Maxime; Longbottom, David; Svoboda, Pavel; Pohl, Jan; Duncan, W Colin; Rae, Michael T; Barlow, Peter G

    2016-09-01

    Cationic host defence peptides (CHDP; also known as antimicrobial peptides) are key components of the immune response in the female reproductive tract. The role of the placental trophoblast in ovine host defence remains poorly understood. This study characterises expression of genes for cathelicidin and defensin peptides in primary ovine placental tissues, the ovine trophoblast cell line (AH-1) and in response to the TLR-4 ligand LPS, the abortifacient organism Waddlia chondrophila and 1α,25-dihydroxyvitamin D3. Using RT-PCR, expression of the CHDP SMAP-29, sBD-1 and sBD-2 was assessed in the AH-1 cell line in response to LPS, 1α,25-dihydroxyvitamin D3 exposure (a known stimulator of cathelicidin gene expression), or W. chondrophila infection. Expression of cathelicidin in the trophoblast compartment of the ovine placenta and in the ovine trophoblast cell line (AH-1) was also established. AH-1 cells did not upregulate expression of CHDP in response to LPS, but sBD-1 and sBD-2 expression was significantly increased in response to W. chondrophila infection. SMAP-29 expression was not altered by in vitro exposure to 1α,25-dihydroxyvitamin D3. This study demonstrates that the ovine trophoblast expresses cathelicidins, but does not upregulate expression of CHDP in response to LPS. Ovine trophoblasts are shown to differentially regulate expression of CHDP and lack a demonstrable vitamin D-mediated cathelicidin response. PMID:27348190

  14. Differential expression of antimicrobial peptides in active and latent tuberculosis and its relationship with diabetes mellitus.

    PubMed

    Gonzalez-Curiel, Irma; Castañeda-Delgado, Julio; Lopez-Lopez, Nallely; Araujo, Zaida; Hernandez-Pando, Rogelio; Gandara-Jasso, Benjamin; Macias-Segura, Noe; Enciso-Moreno, Antonio; Rivas-Santiago, Bruno

    2011-08-01

    Tuberculosis (TB) is one of the most important infectious diseases, causing 1.8 million deaths annually worldwide. This problem has increased because of the association with human immmunodeficiency virus and diabetes mellitus type 2, mainly in developing countries. In the past few years it has been highlighted the significance of antimicrobial peptides in the immunopathogenesis of TB ex vivo and in experimental models studies. In this study we analyzed the expression of CAMP, DEFA1, DEFB4, and DEFB103A in patients with latent TB and progressive TB with and without comorbidity with diabetes mellitus type 2. Antimicrobial peptide gene expression increased during progressive TB, which could be used as a biomarker for reactivation. By contrast, patients with diabetes mellitus type 2 have lower antimicrobial peptides gene expression, suggesting that the lack of its proper production in these patients contribute to enhance the risk for TB reactivation.

  15. Regulation and expression of Lcr plasmid-mediated peptides in pesticinogenic Yersinia pestis

    SciTech Connect

    Sample, A.K.

    1987-01-01

    It is shown in this thesis that cells of Lcr/sup +/, Pst/sup -/ Y. pestis KIM are able to express Yops at levels comparable to that of Lcr/sup +/ Yersinia pseudotuberculosis. Pulse-chase radiolabeling with /sup 35/S-methionine was used to demonstrate that Lcr/sup +/, Pst/sup +/ Y. pestis synthesized at least 11 distinct peptides during the low calcium response and that seven of the labeled peptides were rapidly degraded. These seven peptides were stably expressed in Lcr/sup +/, Pst/sup -/ Y. pestis and were of identical molecular weights as the Yops expressed by that strain. Radiolabeled fragments of low molecular weight accumulated in the extracellular medium of Pst/sup +/ cultures and were assumed to be stable degradation fragments derived from Yops. It was also shown that the set of stable peptides, including V antigen, were made during restriction by both Pst/sup +/ and Pst/sup -/ Y. pestis KIM and were located primarily within the cytoplasm. Those radiolabeled peptides which underwent proteolytic degradation in Pst/sup +/ Y. pestis were localized to the outer membrane and extracellular medium in the Pst/sup -/ strain. It is concluded that the failure of Lcr/sup +/, Pst/sup +/ Y. pestis to express Yops is the result of post-translational degradation and is not a block in the synthesis of Yops.

  16. Unifying expression scale for peptide hydrophobicity in proteomic reversed phase high-pressure liquid chromatography experiments.

    PubMed

    Grigoryan, Marine; Shamshurin, Dmitry; Spicer, Victor; Krokhin, Oleg V

    2013-11-19

    As an initial step in our efforts to unify the expression of peptide retention times in proteomic liquid chromatography-mass spectrometry (LC-MS) experiments, we aligned the chromatographic properties of a number of peptide retention standards against a collection of peptides commonly observed in proteomic experiments. The standard peptide mixtures and tryptic digests of samples of different origins were separated under the identical chromatographic condition most commonly employed in proteomics: 100 Å C18 sorbent with 0.1% formic acid as an ion-pairing modifier. Following our original approach (Krokhin, O. V.; Spicer, V. Anal. Chem. 2009, 81, 9522-9530) the retention characteristics of these standards and collection of tryptic peptides were mapped into hydrophobicity index (HI) or acetonitrile percentage units. This scale allows for direct visualization of the chromatographic outcome of LC-MS acquisitions, monitors the performance of the gradient LC system, and simplifies method development and interlaboratory data alignment. Wide adoption of this approach would significantly aid understanding the basic principles of gradient peptide RP-HPLC and solidify our collective efforts in acquiring confident peptide retention libraries, a key component in the development of targeted proteomic approaches.

  17. Cloning, expression, and purification of a new antimicrobial peptide gene from Musca domestica larva.

    PubMed

    Pei, Zhihua; Sun, Xiaoning; Tang, Yan; Wang, Kai; Gao, Yunhang; Ma, Hongxia

    2014-10-01

    Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram- bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.

  18. Trefoil peptide gene expression in small intestinal Crohn's disease and dietary adaptation.

    PubMed

    Poulsom, R; Chinery, R; Sarraf, C; Van Noorden, S; Stamp, G W; Lalani, E N; Elia, G; Wright, N A

    1993-01-01

    We examined the patterns of trefoil peptide gene expression in the ulcer-associated cell lineage (UACL) and mucosa adjacent to Crohn's disease in humans and during gastrointestinal adaptation to enteral feeding in rats. In the UACL, human spasmolytic polypeptide (hSP) mRNA and peptide are present in the acinar and proximal duct cells, whereas pS2 mRNA and peptide are found in the distal duct cells and in the surface cells. In mucosa adjacent to UACL, pS2 mRNA and peptide are expressed ectopically by goblet cells and neuroendocrine cells. Intestinal crypts associated with the UACL showed marked neuroendocrine cell hyperplasia. Ultrastructural immunolocalization showed pS2 to be copackaged in the mucous cell and neuroendocrine granules. The copackaging of a secretory protein in both mucous and neuroendocrine granules, which have different functions, is unusual and indicates an important role for pS2 in the secretory process itself or as a ligand delivered to its receptor via multiple routes. We also cloned the newest trefoil peptide, intestinal trefoil factor (ITF), from human and rat intestinal mucosa. Using in situ hybridization we demonstrated its synthesis by normal rat intestinal goblet cells. RNAse protection analysis revealed that the level of mRNA for rat ITF in small and large intestine was affected by the process of enteral feeding. We conclude that trefoil peptides are widely distributed in the intestine in human inflammatory bowel disease and are of considerable potential functional importance.

  19. [Expression of prostate stem cell antigen (PSCA) and selection of its specific binding peptide].

    PubMed

    Hou, Li-Hua; Du, Yong; Zhang, Xiao-Peng; An, Xiao-Ping; Chen, Wei

    2004-09-01

    Prostate stem cell antigen (PSCA), a homologue of the Ly-6/Thy-1 family of cell surface antigen, is expressed by a majority of human prostate cancers and is a promising target for prostate cancer immunotherapy. To obtain the specific peptide binding with PSCA for targeted immunotherapy, PSCA gene was obtained by RT-PCR from human prostate cancer cell line DU145 and the transcated PSCA (tPSCA) gene was cloned into vector pQE30 for soluble expression in E. coli. The identity of recombinant tPSCA was confirmed through ELISA and western blot by use of anti-PSCA monoclonal antibody. Then the 12-peptide phage display library was screened with the purified tPSCA protein for its specific binding peptide through 3 rounds panning. For identifying the peptide's specificity, the peptide was coupled with EGFP (enhanced green fluorecent protein) by recombinant DNA technology and the recombinant coupled protein was termed 11-EGFP. The binding specificity with tPSCA of 11-EGFP was further confirmed by ELISA and competitive inhibition experiment. Flow cytometry demonstrated its binding specificity with cell line DU145. In conclusion, a 12-amino-acid peptide which could bind with PSCA specifically was found and it may be a potential tool for targeted immunotherapy of prostate carcinoma. PMID:15973992

  20. Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

    PubMed

    Liutkus, Mantas; Fraser, Samuel A; Caron, Karine; Stigers, Dannon J; Easton, Christopher J

    2016-05-17

    Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis. PMID:26918308

  1. AgRP Neurons Control Systemic Insulin Sensitivity via Myostatin Expression in Brown Adipose Tissue.

    PubMed

    Steculorum, Sophie M; Ruud, Johan; Karakasilioti, Ismene; Backes, Heiko; Engström Ruud, Linda; Timper, Katharina; Hess, Martin E; Tsaousidou, Eva; Mauer, Jan; Vogt, Merly C; Paeger, Lars; Bremser, Stephan; Klein, Andreas C; Morgan, Donald A; Frommolt, Peter; Brinkkötter, Paul T; Hammerschmidt, Philipp; Benzing, Thomas; Rahmouni, Kamal; Wunderlich, F Thomas; Kloppenburg, Peter; Brüning, Jens C

    2016-03-24

    Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis. PMID:27015310

  2. Expression profile of hypothalamic neuropeptides in chicken lines selected for high or low residual feed intake.

    PubMed

    Sintubin, P; Greene, E; Collin, A; Bordas, A; Zerjal, T; Tesseraud, S; Buyse, J; Dridi, S

    2014-08-01

    The R(+) and R(-) chicken lines have been divergently selected for high (R(+)) or low (R(-)) residual feed intake. For the same body weight and egg production, the R(+) chickens consume 40% more food than their counterparts R(-) lines. In the present study we sought to determine the hypothalamic expression profile of feeding-related neuropeptides in these lines maintained under fed or food-deprived conditions. In the fed condition, the suppressor of cytokine signaling 3 (SOCS3) was 17-fold lower (P<0.05) and the ghrelin receptor was 7-fold higher (P<0.05) in R(+) compared to R(-) chicken lines. The hypothalamic expression of the other studied genes remained unchanged between the two lines. In the fasted state, orexigenic neuropeptide Y and agouti-related peptide were more responsive, with higher significant levels in the R(+) compared to R(-) chickens, while no significant differences were seen for the anorexigenic neuropeptides pro-opiomelanocortin and corticotropin releasing hormone. Interestingly, C-reactive protein, adiponectin receptor 1 and ghrelin receptor gene expression were significantly higher (12-, 2- and 3-folds, respectively), however ghrelin and melanocortin 5 receptor mRNA levels were lower (4- and 2-folds, P=0.05 and P=0.03, respectively) in R(+) compared to R(-) animals. We identified several key feeding-related genes that are differently expressed in the hypothalamus of R(+) and R(-) chickens and that might explain the difference in feed intake observed between the two lines.

  3. Characterization and Recombinant Expression of Terebrid Venom Peptide from Terebra guttata.

    PubMed

    Moon, John; Gorson, Juliette; Wright, Mary Elizabeth; Yee, Laurel; Khawaja, Samer; Shin, Hye Young; Karma, Yasmine; Musunri, Rajeeva Lochan; Yun, Michelle; Holford, Mande

    2016-03-03

    Venom peptides found in terebrid snails expand the toolbox of active compounds that can be applied to investigate cellular physiology and can be further developed as future therapeutics. However, unlike other predatory organisms, such as snakes, terebrids produce very small quantities of venom, making it difficult to obtain sufficient amounts for biochemical characterization. Here, we describe the first recombinant expression and characterization of terebrid peptide, teretoxin Tgu6.1, from Terebra guttata. Tgu6.1 is a novel forty-four amino acid teretoxin peptide with a VI/VII cysteine framework (C-C-CC-C-C) similar to O, M and I conotoxin superfamilies. A ligation-independent cloning strategy with an ompT protease deficient strain of E. coli was employed to recombinantly produce Tgu6.1. Thioredoxin was introduced in the plasmid to combat disulfide folding and solubility issues. Specifically Histidine-6 tag and Ni-NTA affinity chromatography were applied as a purification method, and enterokinase was used as a specific cleavage protease to effectively produce high yields of folded Tgu6.1 without extra residues to the primary sequence. The recombinantly-expressed Tgu6.1 peptide was bioactive, displaying a paralytic effect when injected into a Nereis virens polychaete bioassay. The recombinant strategy described to express Tgu6.1 can be applied to produce high yields of other disulfide-rich peptides.

  4. Characterization and Recombinant Expression of Terebrid Venom Peptide from Terebra guttata

    PubMed Central

    Moon, John; Gorson, Juliette; Wright, Mary Elizabeth; Yee, Laurel; Khawaja, Samer; Shin, Hye Young; Karma, Yasmine; Musunri, Rajeeva Lochan; Yun, Michelle; Holford, Mande

    2016-01-01

    Venom peptides found in terebrid snails expand the toolbox of active compounds that can be applied to investigate cellular physiology and can be further developed as future therapeutics. However, unlike other predatory organisms, such as snakes, terebrids produce very small quantities of venom, making it difficult to obtain sufficient amounts for biochemical characterization. Here, we describe the first recombinant expression and characterization of terebrid peptide, teretoxin Tgu6.1, from Terebra guttata. Tgu6.1 is a novel forty-four amino acid teretoxin peptide with a VI/VII cysteine framework (C–C–CC–C–C) similar to O, M and I conotoxin superfamilies. A ligation-independent cloning strategy with an ompT protease deficient strain of E. coli was employed to recombinantly produce Tgu6.1. Thioredoxin was introduced in the plasmid to combat disulfide folding and solubility issues. Specifically Histidine-6 tag and Ni-NTA affinity chromatography were applied as a purification method, and enterokinase was used as a specific cleavage protease to effectively produce high yields of folded Tgu6.1 without extra residues to the primary sequence. The recombinantly-expressed Tgu6.1 peptide was bioactive, displaying a paralytic effect when injected into a Nereis virens polychaete bioassay. The recombinant strategy described to express Tgu6.1 can be applied to produce high yields of other disulfide-rich peptides. PMID:26950153

  5. Transfected human neuropeptide Y cDNA expression in mouse pituitary cells. Inducible high expression, peptide characterization, and secretion.

    PubMed

    Dickerson, I M; Dixon, J E; Mains, R E

    1987-10-01

    An expression vector was constructed that placed the cDNA for human neuropeptide Y (NPY) under the control of the mouse metallothionein promoter and was used to transfect the AtT-20 mouse anterior pituitary corticotrope cell line. AtT-20 cells normally process the pro-ACTH/endorphin precursor but do not produce detectable levels of NPY. The resulting AtT-20/NPY cell line (Mt.NPY1a) was used to study the ability of the corticotrope cells to synthesize, process, and secrete the foreign proNPY-related peptide products. The stable cell line created contains approximately 40 copies of proNPY cDNA per cell. NPY mRNA levels and proNPY synthesis were increased at least 35-fold when maximally induced with cadmium; proNPY synthesis was also induced by glucocorticoids. Upon induction the NPY secretion rate was equimolar to that of the endogenous peptides. ProNPY, NPY, and the COOH-terminal peptide produced by this cell line had molecular weight and amino acid-labeling pattern predicted from cDNA sequence data and from previous isolation of NPY-related molecules from NPY-producing cells. The structures of secreted proNPY, NPY, and COOH-terminal peptide, as well as determination of the site of proteolytic cleavage between NPY and the COOH-terminal peptide, were determined by tryptic mapping and Edman degradation of secreted biosynthetically labeled peptide products. The proNPY molecule appears to be processed in the same pathway responsible for cleavage of the endogenous pro-ACTH/endorphin precursor. Secretion of proNPY-derived peptides paralleled secretion of endogenous pro-ACTH/endorphin-derived products, under both basal and stimulated conditions. With induction proNPY expression there is a dose-dependent inhibition of both proNPY and pro-ACTH/endorphin proteolytic processing.

  6. Transient expression of somatostatin messenger RNA and peptide in the hypoglossal nucleus of the neonatal rat.

    PubMed

    Seroogy, K B; Bayliss, D A; Szymeczek, C L; Hökfelt, T; Millhorn, D E

    1991-06-21

    The postnatal developmental expression of somatostatin mRNA and peptide in the rat hypoglossal nucleus was analyzed using immunocytochemical and in situ hybridization techniques. Both the neuropeptide and its cognate mRNA were found to be transiently present within a subpopulation of hypoglossal motoneurons during the neonatal period. At the day of birth, a large population of perikarya situated in caudal, ventral regions of the hypoglossal nucleus expressed somatostatin. By postnatal day 7, the number of hypoglossal somata which expressed somatostatin had diminished considerably, and by 2 weeks postnatal, only few such cell bodies were found. By 3-4 weeks postnatal, somatostatin peptide- and mRNA-containing hypoglossal motoneurons were rarely observed, and in the adult, they were never detected, despite the use of colchicine. A double-labeling co-localization technique was used to demonstrate that somatostatin, when present perinatally, always coexisted with calcitonin gene-related peptide in hypoglossal motoneurons. The latter peptide, in contrast to somatostatin, was expressed in large numbers of somata throughout the entire hypoglossal nucleus and persisted within the motoneurons throughout development into adulthood. These results demonstrate that somatostatin is transiently expressed in motoneurons of the caudal, ventral tier of the hypoglossal nucleus in the neonatal rat. The developmental disappearance of somatostatin is most likely not due to cell death; hypoglossal somata continue to express calcitonin gene-related peptide, with which somatostatin coexisted perinatally, a high levels throughout development. Thus, it appears that the regulation of somatostatin expression in hypoglossal neurons occurs at the level of gene transcription or mRNA stability/degradation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1680035

  7. Glucocorticoids are required for meal-induced changes in the expression of hypothalamic neuropeptides.

    PubMed

    Uchoa, Ernane Torres; Silva, Lilian Eslaine C M; de Castro, Margaret; Antunes-Rodrigues, Jose; Elias, Lucila L K

    2012-06-01

    Glucocorticoid deficiency is associated with a decrease of food intake. Orexigenic peptides, neuropeptide Y (NPY) and agouti related protein (AgRP), and the anorexigenic peptide proopiomelanocortin (POMC), expressed in the arcuate nucleus of the hypothalamus (ARC), are regulated by meal-induced signals. Orexigenic neuropeptides, melanin-concentrating hormone (MCH) and orexin, expressed in the lateral hypothalamic area (LHA), also control food intake. Thus, the present study was designed to test the hypothesis that glucocorticoids are required for changes in the expression of hypothalamic neuropeptides induced by feeding. Male Wistar rats (230-280 g) were subjected to ADX or sham surgery. ADX animals received 0.9% NaCl in the drinking water, and half of them received corticosterone in the drinking water (B: 25 mg/L, ADX+B). Six days after surgery, animals were fasted for 16 h and they were decapitated before or 2 h after refeeding for brain tissue and blood collections. Adrenalectomy decreased NPY/AgRP and POMC expression in the ARC in fasted and refed animals, respectively. Refeeding decreased NPY/AgRP and increased POMC mRNA expression in the ARC of sham and ADX+B groups, with no effects in ADX animals. The expression of MCH and orexin mRNA expression in the LHA was increased in ADX and ADX+B groups in fasted condition, however there was no effect of refeeding on the expression of MCH and orexin in the LHA in the three experimental groups. Refeeding increased plasma leptin and insulin levels in sham and ADX+B animals, with no changes in leptin concentrations in ADX group, and insulin response to feeding was lower in this group. Taken together, these data demonstrated that circulating glucocorticoids are required for meal-induced changes in NPY, AgRP and POMC mRNA expression in the ARC. The lower leptin and insulin responses to feeding may contribute to the altered hypothalamic neuropeptide expression after adrenalectomy.

  8. Peptide inhibition of constitutively activated epithelial Na(+) channels expressed in Xenopus oocytes.

    PubMed

    Ji, H L; Fuller, C M; Benos, D J

    1999-12-31

    The hypothesis that 30-amino acid peptides corresponding to the C-terminal portion of the beta- and/or gamma-rat epithelial sodium channel (rENaC) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (alphabetagamma-rENaC), truncated Liddle's mutants (alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC), and point mutants (alphabeta(Y)gamma-, alphabetagamma(Y)-rENaC) expressed in Xenopus oocytes. The chord conductances of alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC were 2- or 3-fold greater than for wt alphabetagamma-rENaC. Introduction of peptides into oocytes expressing alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na(+) conductances, with apparent dissociation constants (K(d)) ranging from 1700 to 160 microM, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt alphabetagamma-rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as that of wild type (alphabetagamma-). The single channel activities (N.P(o)) of the mutants were approximately 2.2-2.6-fold greater than wt alphabetagamma-rENaC (1.08 +/- 0.24, n = 7) and were reduced to 1.09 +/- 0.17 by 100 microM peptide mixture (n = 9). The peptides were without effect on the single channel properties of either wt or single-point mutants of rENaC. Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (alphabeta(T)gamma(T)) expressed in Xenopus oocytes but not the single-point mutants (alphabeta(Y)gamma or alphabetagamma(Y)). Moreover, the blocking effect of both peptides in combination on alphabeta(T)gamma(T)-rENaC was synergistic. PMID:10608827

  9. Transgenic tobacco expressing a modified spider peptide inhibits the growth of plant pathogens and insect larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene encoding lycotoxin I, an amphipathic pore-forming peptide, was modified to increase oral toxicity to insects. One of the most active modified genes was then constitutively expressed in tobacco (Nicotiana tabacum) and transformants were evaluated for insect and disease resistance. Pathogenic...

  10. Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish.

    PubMed

    Douglas, Susan E; Gallant, Jeffrey W; Liebscher, Ryan S; Dacanay, Andrew; Tsoi, Stephen C M

    2003-01-01

    Antimicrobial peptides play a crucial role as the first line of defense against invading pathogens. Several types of antimicrobial peptides have been isolated from fish, mostly of the cationic alpha-helical variety. Here, we present the cDNA sequences of five highly disulphide-bonded hepcidin-like peptides from winter flounder, Pseudopleuronectes americanus (Walbaum) and two from Atlantic salmon, Salmo salar (L.). These hepcidin-like molecules consist of a 24 amino acid signal peptide and an acidic propiece of 38-40 amino acids in addition to the mature processed peptide of 19-27 amino acids. Exhaustive data mining of GenBank with these sequences revealed that similar peptides are encoded in the genomes of Japanese flounder, rainbow trout, hybrid striped bass and medaka, indicating that they are widespread among fish. Southern hybridization analysis suggests that closely related hepcidin-like genes are present in other flatfish species, and that they exist as a multigene family clustered on the winter flounder genome. Hepcidin variants are differentially expressed during bacterial challenge, during larval development of P. americanus and in different tissues of adult fish.

  11. Inhibition of NF-kappaB activity by plasmid expressed alphaMSH peptide.

    PubMed

    Etemad-Moghadam, Bijan; Chen, Hongmin; Yin, Peng; Aziz, Nazneen; Hedley, Mary Lynne

    2002-04-01

    Alpha-Melanocyte Stimulating Hormone (alphaMSH) is a neuroimmunomodulatory peptide with remarkable anti-inflammatory properties. Daily or twice daily administration of the peptide reduces the symptoms of several inflammatory animal disease models and the peptide has demonstrated safety in human trials. Unfortunately, the pharmacokinetics of peptide delivery are not favorable from the pharmaceutical perspective. For this reason, plasmid-based vectors were created that constitutively express the immunomodulatory peptide. The fusion constructs encode the 13 amino acids of alphaMSH in frame with the first domain of serum albumin, separated by a linker and furin cleavage sites. The fusion proteins were expressed and processed in human fetal kidney (293) cells. Supernatant from B16/F10 cells transfected with the constructs stimulated secretion of melanin from melanocytes. Furthermore, transfected cytoskeletal muscle (Sol8) cells secreted bioactive alphaMSH that reduced NF-kappaB-mediated transcriptional activation of a luciferase reporter gene. The activity of these vectors provides tools and the impetus for testing the constructs in several animal models of chronic inflammation. PMID:11960637

  12. Diurnal changes in hypothalamic neuropeptide and SOCS-3 expression: effects of lactation and relationship with serum leptin and food intake.

    PubMed

    Denis, R G P; Bing, C; Brocklehurst, S; Harrold, J A; Vernon, R G; Williams, G

    2004-10-01

    Rats normally eat about 85% of their food at night. Lactation increases food intake 3- to 4-fold, but the diurnal pattern of food intake persists. The mechanisms responsible for the diurnal and lactation-induced changes in food intake are still unresolved, hence we have further investigated the possible roles of serum leptin and hypothalamic expression of neuropeptide Y (NPY), agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) in rats. Suppressor of cytokine signalling-3 (SOCS-3) acts as a feedback inhibitor of leptin signalling in the hypothalamus, hence changes in expression of SOCS-3 were also investigated. Changes in expression of NPY, AgRP or POMC alone could not account for the diurnal changes in intake and their alteration by lactation. However, there were increased AgRP mRNA:POMC mRNA ratios at night and also during lactation, which were very similar to estimated changes in food intake. Such changes in expression may result in dominance of the orexigenic AgRP peptide over the appetite-suppressing POMC-derived peptides, and so could contribute to the hyperphagia in these states. Diurnal and lactation-related changes in the AgRP mRNA:POMC mRNA ratio and food intake are not due to changes in leptin alone. However, hypoleptinaemia, possibly through increased expression of NPY, may contribute to the hyperphagia of lactation. In the dark, expression of SOCS-3 was decreased in non-lactating rats; lactation decreased SOCS-3 expression in both light and dark phases. However, such changes are likely to enhance the ability of leptin-responsive neurones to transmit the leptin signal, and so are unlikely to contribute to either the nocturnal increase in appetite or the hyperphagia of lactation.

  13. Induction of Cationic Chicken Liver-Expressed Antimicrobial Peptide 2 in Response to Salmonella enterica Infection

    PubMed Central

    Townes, Claire L.; Michailidis, Georgios; Nile, Christopher J.; Hall, Judith

    2004-01-01

    Cationic antimicrobial peptides constitute part of the innate immune system and provide an essential role in the defense against infection. At present there is a paucity of information regarding the antimicrobial profile of the chicken (Gallus gallus). Using in silico studies, an expressed sequence tag (EST) clone was identified which encodes a novel cationic antimicrobial peptide, chicken liver-expressed antimicrobial peptide 2 (cLEAP-2). The predicted amino acid sequence composed a prepropeptide, and the active peptide contained four conserved cysteine amino acids. The gene was localized to chromosome 13, and analysis of the genome revealed three exons separated by two introns. The cLEAP-2 gene was expressed in a number of chicken epithelial tissues including the small intestine, liver, lung, and kidney. Northern analysis identified liver-specific cLEAP-2 splice variants, suggesting some degree of tissue-specific regulation. To investigate whether cLEAP-2 expression was constitutive or induced in response to microbial infection, 4-day-old birds were orally infected with Salmonella. Analyses of cLEAP-2 expression by semiquantitative reverse transcription-PCR indicated that cLEAP-2 mRNA was upregulated significantly in the small intestinal tissues and the liver, indicative of direct and systemic responses. The antimicrobial activity of cLEAP-2 against Salmonella was analyzed in vitro with a time-kill assay and recombinant cLEAP-2. Interestingly Salmonella enterica serovar Typhimurium SL1344 showed increased susceptibility to the active cationic peptide (amino acids 37 to 76) compared to S. enterica serovar Typhimurium C5 and Salmonella enteritidis. Taken together, these data suggest that cationic cLEAP-2 is part of the innate host defense mechanisms of the chicken. PMID:15557621

  14. High-Affinity PEGylated Polyacridine Peptide Polyplexes Mediate Potent In Vivo Gene Expression

    PubMed Central

    Kizzire, Koby; Khargharia, Sanjib; Rice, Kevin G.

    2012-01-01

    PEGylated polyacridine peptides bind to plasmid DNA with high affinity to form unique polyplexes that possess a long circulatory half-life and are hydrodynamically (HD)-stimulated to produce efficient gene expression in the liver of mice. We previously demonstrated that (Acr-Lys)6-Cys-PEG5kDa stabilizes a 1 μg pGL3 dose for up to 1 hr in the circulation, resulting in HD-stimulated (saline only) gene expression in the liver, equivalent in magnitude to direct-HD dosing of 1 μg of pGL3 (Fernandez C.A. et al. Gene Therapy 2011). In the present study we report that increasing the spacing of Acr with either 4 or 5 Lys residues, dramatically increases the stability of PEGylated polyacridine peptide polyplexes in the circulation allowing maximal HD-stimulated expression for up to 5 hrs post-DNA administration. Co-administration of a decoy dose of 9 μg of non-expressing DNA polyplex with 1 μg of pGL3 polyplex further extended the HD-stimulated expression to 9 hrs. This structure-activity relationship study defines the PEGylated polyacridine peptide requirements for maintaining fully transfection competent plasmid DNA in the circulation for 5 hrs and provides an understanding as to why polyplexes or lipoplexes prepared with PEI, chitosan or Lipofectamine are inactive within 5 min following i.v. dosing. PMID:22786534

  15. Design, recombinant expression, and antibacterial activity of the cecropins-melittin hybrid antimicrobial peptides.

    PubMed

    Cao, Yu; Yu, Rong Qing; Liu, Yi; Zhou, Huo Xiang; Song, Ling Ling; Cao, Yi; Qiao, Dai Rong

    2010-09-01

    In order to evaluate their antibacterial activities and toxicities, the cecropins-melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp's 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect. PMID:20111863

  16. Airway Epithelial Cells are the Site of Expression of a Mammalian Antimicrobial Peptide Gene

    NASA Astrophysics Data System (ADS)

    Diamond, Gill; Jones, Douglas E.; Bevins, Charles L.

    1993-05-01

    We previously reported the isolation and characterization of a broad-spectrum antimicrobial peptide from the bovine tracheal mucosa, which we called tracheal antimicrobial peptide (TAP). We now show the TAP gene is expressed throughout the adult conducting airway, from nasal to bronchiolar tissue, but not in tissues other than airway mucosa, as determined by Northern blot analysis. In situ hybridization of airway sections localizes TAP mRNA to columnar cells of the pseudostratified epithelium. We report the structural organization of the TAP gene and show that TAP is a member of a large family of related sequences with high nucleotide identity in the 5'exon. The data support the hypothesis that antimicrobial peptides contribute to host defense of the respiratory tract.

  17. Modulation of CD59 expression by restrictive silencer factor-derived peptides in cancer immunotherapy for neuroblastoma.

    PubMed

    Donev, Rossen M; Gray, Lisa C; Sivasankar, Baalasubramanian; Hughes, Timothy R; van den Berg, Carmen W; Morgan, B Paul

    2008-07-15

    Tumor cells escape clearance by complement by abundantly expressing CD59 and other membrane complement regulators. Existing strategies for blocking/knocking down these regulators can contribute to tumor immunoclearance in vitro; however, there are numerous difficulties restricting their use in vivo. Here, we report a new strategy for suppression of CD59 expression in neuroblastoma using peptides that target regulators of CD59 expression. We identified the neural-restrictive silencer factor (REST) as a target for modulation of CD59 expression in neuroblastoma. We next designed plasmids that encoded peptides comprising different DNA-binding domains of REST and transfected them into neuroblastoma cell lines. These peptides suppressed CD59 expression, sensitizing neuroblastoma to complement-mediated killing triggered by anti-GD2 therapeutic monoclonal antibody. These CD59-modulating peptides might be effective therapeutic adjuvants to therapeutic monoclonal antibodies used for treatment of neuroblastoma and other cancer types sharing the same mechanism for regulation of CD59 expression.

  18. Prokaryotic expression and antimicrobial mechanism of XPF-St7-derived α-helical peptides.

    PubMed

    Yi, Tonghui; Huang, Yibing; Chen, Yuxin

    2015-01-01

    XPF-St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α-helical segment of XPF-St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram-negative and Gram-positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6-fold and 6.7-fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane.

  19. The gut microbiota reduces leptin sensitivity and the expression of the obesity-suppressing neuropeptides proglucagon (Gcg) and brain-derived neurotrophic factor (Bdnf) in the central nervous system.

    PubMed

    Schéle, Erik; Grahnemo, Louise; Anesten, Fredrik; Hallén, Anna; Bäckhed, Fredrik; Jansson, John-Olov

    2013-10-01

    The gut microbiota contributes to fat mass and the susceptibility to obesity. However, the underlying mechanisms are not completely understood. To investigate whether the gut microbiota affects hypothalamic and brainstem body fat-regulating circuits, we compared gene expression of food intake-regulating neuropeptides between germ-free and conventionally raised (CONV-R) mice. We found that CONV-R mice had decreased expression of the antiobesity neuropeptide glucagon-like peptide-1 (GLP-1) precursor proglucagon (Gcg) in the brainstem. Moreover, in both the hypothalamus and the brainstem, CONV-R mice had decreased expression of the antiobesity neuropeptide brain-derived neurotrophic factor (Bdnf). CONV-R mice had reduced expression of the pro-obesity peptides neuropeptide-Y (Npy) and agouti-related protein (Agrp), and increased expression of the antiobesity peptides proopiomelanocortin (Pomc) and cocaine- and amphetamine-regulated transcript (Cart) in the hypothalamus. The latter changes in neuropeptide expression could be secondary to elevated fat mass in CONV-R mice. Leptin treatment caused less weight reduction and less suppression of orexigenic Npy and Agrp expression in CONV-R mice compared with germ-free mice. The hypothalamic expression of leptin resistance-associated suppressor of cytokine signaling 3 (Socs-3) was increased in CONV-R mice. In conclusion, the gut microbiota reduces the expression of 2 genes coding for body fat-suppressing neuropeptides, Gcg and Bdnf, an alteration that may contribute to fat mass induction by the gut microbiota. Moreover, the presence of body fat-inducing gut microbiota is associated with hypothalamic signs of Socs-3-mediated leptin resistance, which may be linked to failed compensatory body fat reduction.

  20. Tolerance is established in polyclonal CD4(+) T cells by distinct mechanisms, according to self-peptide expression patterns.

    PubMed

    Malhotra, Deepali; Linehan, Jonathan L; Dileepan, Thamotharampillai; Lee, You Jeong; Purtha, Whitney E; Lu, Jennifer V; Nelson, Ryan W; Fife, Brian T; Orr, Harry T; Anderson, Mark S; Hogquist, Kristin A; Jenkins, Marc K

    2016-02-01

    Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however, which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs, which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus, the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms, according to self-peptide expression patterns. PMID:26726812

  1. IL-10 inhibits while calcitriol reestablishes placental antimicrobial peptides gene expression.

    PubMed

    Olmos-Ortiz, Andrea; Noyola-Martínez, Nancy; Barrera, David; Zaga-Clavellina, Verónica; Avila, Euclides; Halhali, Ali; Biruete, Benjamín; Larrea, Fernando; Díaz, Lorenza

    2015-04-01

    IL-10 and calcitriol help to achieve a successful pregnancy by suppressing active maternal immunity; however, these factors exert opposite effects upon microbial infections. In the skin and immune cells, IL-10 downregulates β-defensins while calcitriol induces cathelicidin gene expression in various tissues including placenta. Though, the regulation of human placental β-defensins by IL-10 and calcitriol has not been studied. Therefore, we explored the regulation of these antimicrobial peptides expression in cultured placental cells by calcitriol and IL-10 alone and combined. Real time PCR showed that calcitriol stimulated, while IL-10 inhibited, β-defensins and cathelicidin gene expression (P<0.05). In coincubations studies, calcitriol was able to maintain antimicrobial peptides gene expression above control values, overriding IL-10 inhibitory effects. Calcitriol downregulated endogenous IL-10 secretion. Interestingly, calcitriol and TNF-α cooperatively enhanced β-defensins, while TNF-α reduced basal and calcitriol-stimulated cathelicidin gene expression. In summary, calcitriol and IL-10 exerted opposite effects on antimicrobial peptides expression in the human placenta, suggesting that unbalanced production of IL-10 and calcitriol could be deleterious to innate immune responses during gestation. Our results suggest that calcitriol enhancement of placental defenses involves two mechanisms: (1) downregulation of IL-10 secretion and (2) direct upregulation of β-defensins and cathelicidin gene expression. Considering that IL-10 and calcitriol differentially regulate the innate immune response in the placenta, in the case of an infection, calcitriol might restrict IL-10 permissive actions towards microbial invasion while restrains inflammation, allowing for pregnancy to continue in quiescence. These results strongly advice maternal vitamin D sufficiency during pregnancy.

  2. Milk peptides increase iron solubility in water but do not affect DMT-1 expression in Caco-2 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. Our objectives were to investigate whether these fractions a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells b) enhance iron dialyzability when added in meals. Peptid...

  3. Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes.

    PubMed

    Schulze, W; Frommer, W B; Ward, J M

    1999-03-01

    Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In Nepenthes, insect-derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in Nepenthes pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of Nepenthes. Expression of the ammonium transporter gene NaAMT1 was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene, NaAAP1, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by NaAAP1 may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene NaNTR1 was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem. PMID:10230062

  4. Tryptophan inhibits Proteus vulgaris TnaC leader peptide elongation, activating tna operon expression.

    PubMed

    Cruz-Vera, Luis R; Yang, Rui; Yanofsky, Charles

    2009-11-01

    Expression of the tna operon of Escherichia coli and of Proteus vulgaris is induced by L-tryptophan. In E. coli, tryptophan action is dependent on the presence of several critical residues (underlined) in the newly synthesized TnaC leader peptide, WFNIDXXL/IXXXXP. These residues are conserved in TnaC of P. vulgaris and of other bacterial species. TnaC of P. vulgaris has one additional feature, distinguishing it from TnaC of E. coli; it contains two C-terminal lysine residues following the conserved proline residue. In the present study, we investigated L-tryptophan induction of the P. vulgaris tna operon, transferred on a plasmid into E. coli. Induction was shown to be L-tryptophan dependent; however, the range of induction was less than that observed for the E. coli tna operon. We compared the genetic organization of both operons and predicted similar folding patterns for their respective leader mRNA segments. However, additional analyses revealed that L-tryptophan action in the P. vulgaris tna operon involves inhibition of TnaC elongation, following addition of proline, rather than inhibition of leader peptide termination. Our findings also establish that the conserved residues in TnaC of P. vulgaris are essential for L-tryptophan induction, and for inhibition of peptide elongation. TnaC synthesis is thus an excellent model system for studies of regulation of both peptide termination and peptide elongation, and for studies of ribosome recognition of the features of a nascent peptide. PMID:19767424

  5. GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION

    PubMed Central

    Chao, Celia; Ives, Kirk; Hellmich, Helen L.; Townsend, Courtney M.; Hellmich, Mark R.

    2015-01-01

    Background Breast cancers aberrantly express gastrin-releasing peptide (GRP) hormone and its cognate receptor, gastrin-releasing peptide receptor (GRP-R). Experimental evidence suggests that bombesin (BBS), the pharmacological homologue of GRP, promotes breast cancer growth and progression. The contribution of GRP-R to other poor prognostic indicators in breast cancer, such as the expression of the EGF-R family of growth factors, and hormone insensitivity is unknown. Materials and Methods Two estrogen receptor (ER)-negative breast cancer cell lines were used. MDA-MB-231 overexpress both EGFR and GRPR, whereas SK-BR-3 cells express EGF-R but lack GRP-R. Cellular proliferation was assessed by Coulter counter. Chemotactic migration was performed using Transwell chambers and the migrated cells were quantified. Northern blot and real-time PCR were used to evaluate if pro-angiogenic factor interleukin-8 (IL-8) mRNA expression. Results In MDA-MB-231 cells, GRP-R and EGF-R synergize to regulate cell migration, IL-8 expression, but not cell proliferation. In SK-BR-3 cells, ectopic expression of GRP-R was sufficient to increase migration and IL-8 mRNA. Conclusions These data suggest relevant roles for GRP-R in ER-negative breast cancer progression. Future mechanistic studies to define the molecular role of GRP-R in breast cancer metastasis provide novel targets for the treatment of ER-negative breast cancers. PMID:19631337

  6. Posttranslational processing of endogenous and of baculovirus-expressed human gastrin-releasing peptide precursor.

    PubMed Central

    Lebacq-Verheyden, A M; Kasprzyk, P G; Raum, M G; Van Wyke Coelingh, K; Lebacq, J A; Battey, J F

    1988-01-01

    The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands. Images PMID:3211139

  7. Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding

    PubMed Central

    Chappell, Paul E; Meziane, El Kahina; Harrison, Michael; Magiera, Łukasz; Hermann, Clemens; Mears, Laura; Wrobel, Antoni G; Durant, Charlotte; Nielsen, Lise Lotte; Buus, Søren; Ternette, Nicola; Mwangi, William; Butter, Colin; Nair, Venugopal; Ahyee, Trudy; Duggleby, Richard; Madrigal, Alejandro; Roversi, Pietro; Lea, Susan M; Kaufman, Jim

    2015-01-01

    Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. DOI: http://dx.doi.org/10.7554/eLife.05345.001 PMID:25860507

  8. Aminopeptidase N gene expression and abundance in caprine mammary gland is influenced by circulating plasma peptide.

    PubMed

    Mabjeesh, S J; Gal-Garber, O; Milgram, J; Feuermann, Y; Cohen-Zinder, M; Shamay, A

    2005-06-01

    This study examined the localization and the effect of circulating peptides on the expression of aminopeptidase N (EC 3.4.11.2) in caprine mammary gland. Four lactating goats in mid to late lactation were used in a crossover design and were subjected to 2 dietary treatments. Abomasal infusion of casein hydrolysate was used to increase the concentration of peptide-bound amino acid in the circulation. Samples of mammary gland tissue from each goat were taken by biopsy at the end of each treatment period to measure gene and protein expression of aminopeptidase N in the tissue. There were no measurable effects on feed intake and milk production for any of the treatments. Western blot analysis showed that aminopeptidase N is located on the basolateral side of parenchymal cells and not on the apical membranes. Abomasal infusion of casein hydrolysate caused a marked change in the profile of arterial blood free amino acids and peptide-bound amino acids smaller than 1500 Da. Abundance of aminopeptidase N mRNA and protein increased by 51 and 58%, respectively, in casein hydrolysate-infused goats compared with the control treatment. It was concluded that aminopeptidase N is one candidate actively involved in the mammary gland to support protein synthesis and milk production. In accordance with the nutritional conditions in the current experiment, it is suggested that aminopeptidase N expression is partly controlled by the metabolic requirements of the gland and postabsorptive forms of amino acids in the circulation. PMID:15905436

  9. Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology.

    PubMed

    Holaskova, Edita; Galuszka, Petr; Frebort, Ivo; Oz, M Tufan

    2015-11-01

    Antimicrobial peptides (AMPs) are vital components of the innate immune system of nearly all living organisms. They generally act in the first line of defense against various pathogenic bacteria, parasites, enveloped viruses and fungi. These low molecular mass peptides are considered prospective therapeutic agents due to their broad-spectrum rapid activity, low cytotoxicity to mammalian cells and unique mode of action which hinders emergence of pathogen resistance. In addition to medical use, AMPs can also be employed for development of innovative approaches for plant protection in agriculture. Conferred disease resistance by AMPs might help us surmount losses in yield, quality and safety of agricultural products due to plant pathogens. Heterologous expression in plant-based systems, also called plant molecular farming, offers cost-effective large-scale production which is regarded as one of the most important factors for clinical or agricultural use of AMPs. This review presents various types of AMPs as well as plant-based platforms ranging from cell suspensions to whole plants employed for peptide production. Although AMP production in plants holds great promises for medicine and agriculture, specific technical limitations regarding product yield, function and stability still remain. Additionally, establishment of particular stable expression systems employing plants or plant tissues generally requires extended time scale for platform development compared to certain other heterologous systems. Therefore, fast and promising tools for evaluation of plant-based expression strategies and assessment of function and stability of the heterologously produced AMPs are critical for molecular farming and plant protection.

  10. Comparison of antibody avidity and titre elicited by peptide as a protein conjugate or as expressed in vaccinia.

    PubMed Central

    Lew, A M; Anders, R F; Edwards, S J; Langford, C J

    1988-01-01

    The immunogenicity of a malaria peptide presented in various forms was tested in terms of the quality and quantity of the antibody response in rabbits. Peptide conjugated to a protein carrier, diphtheria toxoid (DT), required strong adjuvants (e.g. muramyl dipeptide, MDP and Freund's adjuvant, FCA) to elicit high levels of antibody with high avidity. Alum was a poor adjuvant, producing the lowest titre and avidity of antibody compared with all the other groups. Peptide expressed in vaccinia (and without carrier) produced intermediate levels of antibody but the avidity of the antibodies produced were comparable to that produced by peptide conjugates given with muramyl dipeptide. PMID:3056855

  11. Nesfatin-1 stimulates cholecystokinin and suppresses peptide YY expression and secretion in mice.

    PubMed

    Ramesh, Naresh; Mortazavi, Sima; Unniappan, Suraj

    2016-03-25

    Nesfatin-1 is an 82 amino acid secreted peptide encoded in the precursor, nucleobindin-2 (NUCB2). It is an insulinotropic anorexigen abundantly expressed in the stomach and hypothalamus. Post-prandial insulin secretion is predominantly regulated by incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Nesfatin-1 was previously reported to modulate GLP-1 and GIP secretion in vitro in an enteroendocrine (STC-1) cell line. Intestine is a source of additional hormones including cholecystokinin (CCK) and peptide YY (PYY) that regulate metabolism. We hypothesized that nesfatin-1 modulates CCK and PYY secretion. Immunofluorescence histochemistry showed NUCB2/nesfatin-1 co-localizing CCK and PYY in the intestinal mucosa of mice. Static incubation of STC-1 cells with nesfatin-1 upregulated both CCK mRNA expression (1 and 10 nM) and secretion (0.1, 1 and 10 nM) at 1 h post-incubation. In contrast, nesfatin-1 treatment for 1 h downregulated PYY mRNA expression (all doses tested) and secretion (0.01 and 0.1 nM) in STC-1 cells. Continuous infusion of nesfatin-1 using osmotic mini-pumps for 12 h upregulated CCK mRNA expression in large intestine, and downregulated PYY mRNA expression in both large and small intestines of male C57BL/6J mice. In these tissues, Western blot analysis found a corresponding increase in CCK and a decrease in PYY content. Collectively, we provide new information on the cell specific localization of NUCB2/nesfatin-1 in the intestinal mucosa, and a novel function for nesfatin-1 in modulating intestinal CCK and PYY expression and secretion in mice.

  12. Milk peptides increase iron dialyzability in water but do not affect DMT-1 expression in Caco-2 cells.

    PubMed

    Argyri, Konstantina; Tako, Elad; Miller, Dennis D; Glahn, Raymond P; Komaitis, Michael; Kapsokefalou, Maria

    2009-02-25

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. The objectives of this study were to investigate whether these fractions (a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells and (b) enhance iron dialyzability when added in meals. Two milk peptide fractions that solubilize iron were isolated by Sephadex G-25 gel filtration of a milk digest. These peptide fractions did not affect relative gene expression of DMT-1 when incubated with Caco-2 cells for 2 or 48 h. Dialyzability was measured after in vitro simulated gastric and pancreatic digestion. Both peptide fractions enhanced the dialyzability of iron from ferric chloride added to PIPES buffer, but had no effect on dialyzability from milk or a vegetable or fruit meal after in vitro simulated gastric and pancreatic digestion. However, dialyzability from milk was enhanced by the addition of a more concentrated lyophilized peptide fraction.

  13. A cell permeable peptide inhibitor of NFAT inhibits macrophage cytokine expression and ameliorates experimental colitis.

    PubMed

    Elloumi, Houda Z; Maharshak, Nitsan; Rao, Kavitha N; Kobayashi, Taku; Ryu, Hyungjin S; Mühlbauer, Marcus; Li, Fengling; Jobin, Christian; Plevy, Scott E

    2012-01-01

    Nuclear factor of activated T cells (NFAT) plays a critical role in the development and function of immune and non-immune cells. Although NFAT is a central transcriptional regulator of T cell cytokines, its role in macrophage specific gene expression is less defined. Previous work from our group demonstrated that NFAT regulates Il12b gene expression in macrophages. Here, we further investigate NFAT function in murine macrophages and determined the effects of a cell permeable NFAT inhibitor peptide 11R-VIVIT on experimental colitis in mice. Treatment of bone marrow derived macrophages (BMDMs) with tacrolimus or 11R-VIVIT significantly inhibited LPS and LPS plus IFN-γ induced IL-12 p40 mRNA and protein expression. IL-12 p70 and IL-23 secretion were also decreased. NFAT nuclear translocation and binding to the IL-12 p40 promoter was reduced by NFAT inhibition. Experiments in BMDMs from IL-10 deficient (Il10(-/-)) mice demonstrate that inhibition of IL-12 expression by 11R-VIVIT was independent of IL-10 expression. To test its therapeutic potential, 11R-VIVIT was administered systemically to Il10(-/-) mice with piroxicam-induced colitis. 11R-VIVIT treated mice demonstrated significant improvement in colitis compared to mice treated with an inactive peptide. Moreover, decreased spontaneous secretion of IL-12 p40 and TNF in supernatants from colon explant cultures was demonstrated. In summary, NFAT, widely recognized for its role in T cell biology, also regulates important innate inflammatory pathways in macrophages. Selective blocking of NFAT via a cell permeable inhibitory peptide is a promising therapeutic strategy for the treatment of inflammatory bowel diseases.

  14. Maternal nicotine exposure during lactation alters hypothalamic neuropeptides expression in the adult rat progeny.

    PubMed

    Younes-Rapozo, Viviane; Moura, Egberto G; Manhães, Alex C; Pinheiro, Cintia R; Santos-Silva, Ana Paula; de Oliveira, Elaine; Lisboa, Patricia C

    2013-08-01

    Maternal exposure to nicotine during lactation causes hyperleptinemia in the pups and, at adulthood, these animals are overweight and hyperleptinemic, while, in their hypothalamus, the leptin signaling pathway is reduced, evidencing a central leptin resistance. Then, we evaluated the expression of pro-opiomelanocortin (POMC), alpha-melanocyte stimulating hormone (α-MSH), cocaine and amphetamine-regulated transcript (CART), neuropeptide Y (NPY), agouti-related peptide (AgRP) and others in different hypothalamic nuclei in order to better understand the mechanisms underlying the obese phenotype observed in these animals at adulthood. On the 2nd postnatal day (P2), dams were subcutaneously implanted with osmotic minipumps releasing nicotine (NIC-6 mg/kg/day) or saline for 14 days. Offspring were killed in P180 and immunohistochemistry and Western blot analysis were carried out. Significance data had p<0.05. Adult NIC offspring showed more intense NPY staining in the paraventricular nucleus (PVN) (+21%) and increased number of POMC-positive cells in the: arcuate nucleus (+33%), as an increase in fiber density of α-MSH in PVN (+85%). However, the number of CART-positive cells was reduced in the PVN (-25%). CRH staining was more intense in NIC offspring (+136%). Orexins and AgRP were not altered. Thus, maternal nicotine exposure changes hypothalamic neuropeptides in the adult progeny that is partially compatible with leptin resistance.

  15. Mice that are resistant to diet-induced weight loss have greater food anticipatory activity and altered melanocortin-3 receptor (MC3R) and dopamine receptor 2 (D2) gene expression.

    PubMed

    Vaanholt, Lobke M; Mitchell, Sharon E; Sinclair, Rachel E; Speakman, John R

    2015-07-01

    Diet-induced weight loss varies considerably between individuals, but the mechanisms driving these individual differences remain largely unknown. Here we investigated whether key neuropeptides involved in the regulation of energy balance or reward systems were differentially expressed in mice that were prone or resistant to caloric restriction (CR) induced weight loss. Mice (n=30 males and n=34 females) were fed 70% of their own baseline ad libitum intake for 25days, after which their brains were collected and expression of various neuropeptides were investigated and compared between the 10 male and 10 female mice that showed the greatest (high weight loss, HWL) or lowest weight loss (LWL) (n=40 in total). HWL mice showed a differential neuropeptide profile to LWL in both sexes, characterised by increased expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), leptin receptor (ObRb), and melanocortin 3 receptor (MC3R) in the arcuate nucleus. No changes in the expression of fat mass and obesity related gene (FTO) or suppressor of cytokine signalling 3 (Socs3) were observed. Levels of dopamine D2 receptor were decreased in the nucleus accumbens in HWL compared to LWL mice. HWL mice showed a stronger increase in food anticipatory activity (FAA) in response to CR than LWL mice. These results indicate that the mice prone to diet-induced weight loss experienced greater hunger, potentially driving their elevated FAA.

  16. Distinct regulation of vasoactive intestinal peptide (VIP) expression at mRNA and peptide levels in human neuroblastoma cells.

    PubMed

    Agoston, D V; Colburn, S; Krajniak, K G; Waschek, J A

    1992-05-25

    Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

  17. Identification and characterization of latency-associated peptide-expressing γδ T cells

    PubMed Central

    Rezende, Rafael M.; da Cunha, Andre P.; Kuhn, Chantal; Rubino, Stephen; M'Hamdi, Hanane; Gabriely, Galina; Vandeventer, Tyler; Liu, Shirong; Cialic, Ron; Pinheiro-Rosa, Natalia; Oliveira, Rafael P.; Gaublomme, Jellert T.; Obholzer, Nikolaus; Kozubek, James; Pochet, Nathalie; Faria, Ana M. C.; Weiner, Howard L.

    2015-01-01

    γδ T cells are a subset of lymphocytes specialized in protecting the host against pathogens and tumours. Here we describe a subset of regulatory γδ T cells that express the latency-associated peptide (LAP), a membrane-bound TGF-β1. Thymic CD27+IFN-γ+CCR9+α4β7+TCRγδ+ cells migrate to the periphery, particularly to Peyer's patches and small intestine lamina propria, where they upregulate LAP, downregulate IFN-γ via ATF-3 expression and acquire a regulatory phenotype. TCRγδ+LAP+ cells express antigen presentation molecules and function as antigen presenting cells that induce CD4+Foxp3+ regulatory T cells, although TCRγδ+LAP+ cells do not themselves express Foxp3. Identification of TCRγδ+LAP+ regulatory cells provides an avenue for understanding immune regulation and biologic processes linked to intestinal function and disease. PMID:26644347

  18. Expression pattern of arenicins—the antimicrobial peptides of polychaete Arenicola marina

    PubMed Central

    Maltseva, Arina L.; Kotenko, Olga N.; Kokryakov, Vladimir N.; Starunov, Viktor V.; Krasnodembskaya, Anna D.

    2014-01-01

    Immune responses of invertebrate animals are mediated through innate mechanisms, among which production of antimicrobial peptides play an important role. Although evolutionary Polychaetes represent an interesting group closely related to a putative common ancestor of other coelomates, their immune mechanisms still remain scarcely investigated. Previously our group has identified arenicins—new antimicrobial peptides of the lugworm Arenicola marina, since then these peptides were thoroughly characterized in terms of their structure and inhibitory potential. In the present study we addressed the question of the physiological functions of arenicins in the lugworm body. Using molecular and immunocytochemical methods we demonstrated that arencins are expressed in the wide range of the lugworm tissues—coelomocytes, body wall, extravasal tissue and the gut. The expression of arenicins is constitutive and does not depend on stimulation of various infectious stimuli. Most intensively arenicins are produced by mature coelomocytes where they function as killing agents inside the phagolysosome. In the gut and the body wall epithelia arenicins are released from producing cells via secretion as they are found both inside the epithelial cells and in the contents of the cuticle. Collectively our study showed that arenicins are found in different body compartments responsible for providing a first line of defense against infections, which implies their important role as key components of both epithelial and systemic branches of host defense. PMID:25566093

  19. Scavenger receptor B protects shrimp from bacteria by enhancing phagocytosis and regulating expression of antimicrobial peptides.

    PubMed

    Bi, Wen-Jie; Li, Dian-Xiang; Xu, Yi-Hui; Xu, Sen; Li, Jing; Zhao, Xiao-Fan; Wang, Jin-Xing

    2015-07-01

    Scavenger receptors (SRs) are involved in innate immunity through recognizing pathogen-associated molecular patterns (PAMPs) and in pathogenesis of diseases through interactions with damage-associated molecular patterns (DAMPs). The roles of SRs in invertebrate innate immunity still need to be elucidated. Here we identify a class B scavenger receptor from kuruma shrimp, Marsupenaeus japonicus, designated MjSR-B1. The recombinant MjSR-B1 agglutinated bacteria in a calcium dependent manner and bound lipopolysaccharide and lipoteichoic acid. After knockdown of MjSR-B1, both the bacterial clearance and phagocytotic ability of M. japonicus against V. anguillarum and S. aureus were impaired, and several phagocytosis related genes were downregulated. The expression levels of antimicrobial peptides were also downregulated. Overexpression of MjSR-B1 led to enhanced bacterial clearance, phagocytosis rate and upregulation of phagocytosis-related and antimicrobial peptide genes. However, overexpression of mutant MjSR-B1ΔC, which lacks the carboxyl tail of MjSR-B1, had none of these effects. Our results indicate that MjSR-B1 can protect shrimp from bacteria by promoting phagocytosis and by enhancing the expression of antimicrobial peptides.

  20. Ethanol enhancement of cocaine- and amphetamine-regulated transcript mRNA and peptide expression in the nucleus accumbens.

    PubMed

    Salinas, Armando; Wilde, Jennifer D; Maldve, Regina E

    2006-04-01

    Cocaine- and amphetamine-regulated transcript (CART) is a peptide neurotransmitter that has been implicated in drug reward and reinforcement. CART mRNA and peptide expression are highly concentrated in several compartments of the mesolimbic reward pathway. Several lines of evidence suggest that CART peptides may contribute to rewarding behaviors and the addiction liability of psychostimulants; however, there are no reports of basic work concerning CART in relation to alcohol and mechanisms of alcohol dependence development. Therefore, in this study we investigated the response of CART transcript and peptide to acute ethanol administration in vivo. Rats were administered ethanol (1 g/kg or 3.5 g/kg, 1 h, ip) and CART expression was measured by RT-PCR in the nucleus accumbens (NAcc). Ethanol (3.5 g/kg) increased CART transcription markedly. The interactions of dopamine on ethanol-induced CART expression were further evaluated pharmacologically using D1 and D2/D3 receptor antagonists. Both SCH 23390 (0.25 mg/kg) or raclopride (0.2 mg/kg) pre-treatment significantly suppressed ethanol-enhancement of CART mRNA transcription. Confocal immunofluorescence microscopy revealed that CART peptide immunoreactivity was also enhanced in both the core and the shell of the NAcc by ethanol administration. These findings demonstrate that CART mRNA and peptide expression are responsive to acute ethanol administrated in vivo and suggests that CART peptides may be important in regulating the rewarding and reinforcing properties of ethanol. PMID:16539670

  1. Effect of opioid receptor antagonism on proopiomelanocortin peptide levels and gene expression in the hypothalamus.

    PubMed

    Markowitz, C E; Berkowitz, K M; Jaffe, S B; Wardlaw, S L

    1992-06-01

    In order to determine how brain beta-endorphin (beta-EP) and its precursor proopiomelanocortin (POMC) adapt to chronic opioid blockade we have examined the effects of treatment with the opioid receptor antagonist naltrexone (NTX) on POMC gene expression and peptide levels in the hypothalamus. Male rats were treated with NTX by daily injection or constant minipump infusion. RNA was isolated from the medial basal hypothalamus (MBH) after an aliquot was removed for peptide RIA and the amount of POMC mRNA was measured by solution hybridization SI nuclease protection assay. beta-EP and several other POMC-derived peptides including alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin-like intermediate lobe peptide (CLIP) or gamma(3)-MSH were measured in the MBH and anterior hypothalamus (AH) by RIA. In an initial experiment POMC peptide levels were measured after 7 days of NTX (4.8 mg/day) infusion. There was a marked fall in the concentrations of beta-EP, alpha-MSH, and CLIP; levels in the MBH declined by more than 60% (P < 0.001). In the next experiment NTX (1 mg) was injected daily and POMC peptides and mRNA were measured after 2 and 5 days of treatment. (beta-EP) and alpha-MSH levels fell progressively in the MBH and AH and were significantly less than those of the controls by 5 days of treatment (P < 0.02). POMC mRNA levels, however, did not change after 2 or 5 days. When NTX was infused for 3 weeks there was a decrease in the concentrations of beta-EP, alpha-MSH, and gamma(3)-MSH in the MBH (P < 0.001). The concentration of POMC mRNA in the MBH, however, was significantly higher in the NTX-treated animals, 0.99 +/- 0.06 pg/mug RNA vs 0.81 +/- 0.05 pg/mug RNA (P < 0.05). Since NTX can affect LH and testosterone release, the study was repeated in castrated rats. POMC peptide levels again fell after 3 weeks of NTX. POMC mRNA levels were higher in the castrated rats than in the intact rats, 1.14 +/- 0.06 pg/mug RNA vs 0.85 +/- 0.09 pg/mug RNA (P < 0

  2. Co-expression of Dorsal and Rel2 Negatively Regulates Antimicrobial Peptide Expression in the Tobacco Hornworm Manduca sexta.

    PubMed

    Zhong, Xue; Rao, Xiang-Jun; Yi, Hui-Yu; Lin, Xin-Yu; Huang, Xiao-Hong; Yu, Xiao-Qiang

    2016-01-01

    Nuclear factor κB (NF-κB) plays an essential role in regulation of innate immunity. In mammals, NF-κB factors can form homodimers and heterodimers to activate gene expression. In insects, three NF-κB factors, Dorsal, Dif and Relish, have been identified to activate antimicrobial peptide (AMP) gene expression. However, it is not clear whether Dorsal (or Dif) and Relish can form heterodimers. Here we report the identification and functional analysis of a Dorsal homologue (MsDorsal) and two Relish short isoforms (MsRel2A and MsRel2B) from the tobacco hornworm, Manduca sexta. Both MsRel2A and MsRel2B contain only a Rel homology domain (RHD) and lack the ankyrin-repeat inhibitory domain. Overexpression of the RHD domains of MsDorsal and MsRel2 in Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells can activate AMP gene promoters from M. sexta and D. melanogaster. We for the first time confirmed the interaction between MsDorsal-RHD and MsRel2-RHD, and suggesting that Dorsal and Rel2 may form heterodimers. More importantly, co-expression of MsDorsal-RHD with MsRel2-RHD suppressed activation of several M. sexta AMP gene promoters. Our results suggest that the short MsRel2 isoforms may form heterodimers with MsDorsal as a novel mechanism to prevent over-activation of antimicrobial peptides. PMID:26847920

  3. Co-expression of Dorsal and Rel2 Negatively Regulates Antimicrobial Peptide Expression in the Tobacco Hornworm Manduca sexta

    PubMed Central

    Zhong, Xue; Rao, Xiang-Jun; Yi, Hui-Yu; Lin, Xin-Yu; Huang, Xiao-Hong; Yu, Xiao-Qiang

    2016-01-01

    Nuclear factor κB (NF-κB) plays an essential role in regulation of innate immunity. In mammals, NF-κB factors can form homodimers and heterodimers to activate gene expression. In insects, three NF-κB factors, Dorsal, Dif and Relish, have been identified to activate antimicrobial peptide (AMP) gene expression. However, it is not clear whether Dorsal (or Dif) and Relish can form heterodimers. Here we report the identification and functional analysis of a Dorsal homologue (MsDorsal) and two Relish short isoforms (MsRel2A and MsRel2B) from the tobacco hornworm, Manduca sexta. Both MsRel2A and MsRel2B contain only a Rel homology domain (RHD) and lack the ankyrin-repeat inhibitory domain. Overexpression of the RHD domains of MsDorsal and MsRel2 in Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells can activate AMP gene promoters from M. sexta and D. melanogaster. We for the first time confirmed the interaction between MsDorsal-RHD and MsRel2-RHD, and suggesting that Dorsal and Rel2 may form heterodimers. More importantly, co-expression of MsDorsal-RHD with MsRel2-RHD suppressed activation of several M. sexta AMP gene promoters. Our results suggest that the short MsRel2 isoforms may form heterodimers with MsDorsal as a novel mechanism to prevent over-activation of antimicrobial peptides. PMID:26847920

  4. Localization of corin and atrial natriuretic peptide expression in human renal segments.

    PubMed

    Dong, Liang; Wang, Hao; Dong, Ningzheng; Zhang, Ce; Xue, Boxin; Wu, Qingyu

    2016-09-01

    Atrial natriuretic peptide (ANP)-mediated natriuretic response is a well-established cardiac endocrine function. Corin is a transmembrane protease that activates ANP in the heart. Corin expression has been detected in non-cardiac tissues including the kidney. Here we examined corin, pro-ANP/ANP and natriuretic peptide receptor-A (NPR-A) expression in human renal segments. By immunostaining and in situ hybridization, we found similar corin, pro-ANP/ANP and NPR-A protein and mRNA expression in human renal segments. The expression was most abundant in the proximal convoluted tubules and the medullary connecting ducts. In the proximal tubules, corin protein was present in the apical membrane region underneath the brush border where the ANP-degrading protease neprilysin was abundant. These results suggest that corin-mediated pro-ANP activation may occur in renal segments and that locally produced ANP may act in an autocrine manner to regulate sodium and water reabsorption in situ Our results also point to the proximal convoluted tubules as a major site for local ANP action. Such a renal corin/ANP autocrine mechanism may differ from the cardiac corin/ANP endocrine mechanism in regulating sodium homoeostasis under physiological and pathological conditions. PMID:27343265

  5. Expression and potential role of the peptide orexin-A in prostate cancer.

    PubMed

    Valiante, Salvatore; Liguori, Giovanna; Tafuri, Simona; Pavone, Luigi Michele; Campese, Roberto; Monaco, Roberto; Iachetta, Giuseppina; Assisi, Loredana; Mirabella, Nicola; Forte, Maurizio; Costagliola, Anna; Vittoria, Alfredo

    2015-09-01

    The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A in human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer.

  6. Chemical genetic analysis reveals the effects of NMU2R on the expression of peptide hormones.

    PubMed

    Fang, Liyan; Zhang, Mancang; Li, Chunxia; Dong, Suzhen; Hu, Yinghe

    2006-08-14

    Neuromedin U 2 receptor (NMU2R) plays important roles for the regulation of food intake and body weight. However, the molecular mechanism underlying the action of NMU2R has not been clearly defined. We have taken chemical genetic approach to examine the involvement of peptides in the regulation of NMU2R effects. A cell-based reporter gene assay has been developed and used for the screening of human NMU2R agonist. Three natural product compounds, EUK2010, EUK2011 and EUK2012, were identified that could activate the reporter gene expression in the cell-based functional assay. Although these compounds showed high EC50 at hundreds micro-molar range, in vitro pharmacological analysis suggested that they were specific agonists for the human NMU2R. The natural compounds could decrease food intake and lead to the reduction of body weight in different animal models. To understand the molecular basis of the NMU2R regulation of food intake and body weight, we examined the expression of a number of key genes in hypothalamus and adipose tissues after oral administration of EUK2010 in mice. Our results demonstrated that the expression levels of a number of neuropeptide genes were altered after the treatment of EUK2010. Interestingly, EUK2010 increased the expression of Leptin in white fat. These results suggested that these peptides may participate in the regulation of NMU2R effects in mice. PMID:16781063

  7. Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation

    PubMed Central

    Rosay, Thibaut; Bazire, Alexis; Diaz, Suraya; Clamens, Thomas; Blier, Anne-Sophie; Mijouin, Lily; Hoffmann, Brice; Sergent, Jacques-Aurélien; Bouffartigues, Emeline; Boireau, Wilfrid; Vieillard, Julien; Hulen, Christian; Dufour, Alain; Harmer, Nicholas J.; Feuilloley, Marc G. J.

    2015-01-01

    ABSTRACT Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23 mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression. PMID:26307165

  8. Prenatal Exposure to Nicotine Stimulates Neurogenesis of Orexigenic Peptide-Expressing Neurons in Hypothalamus and Amygdala

    PubMed Central

    Chang, Guo-Qing; Karatayev, Olga

    2013-01-01

    Animal and clinical studies show that gestational exposure to nicotine increases the propensity of offspring to consume nicotine, but the precise mechanism mediating this behavioral phenomenon is unclear. The present study in Sprague Dawley rats examined the possibility that the orexigenic peptide systems, enkephalin (ENK) and orexin (OX), which are stimulated by nicotine in adult animals and promote consummatory behavior, may be similarly responsive to nicotine's stimulatory effect in utero while having long-term behavioral consequences. The results demonstrated that nicotine exposure during gestation at low doses (0.75 or 1.5 mg/kg/d) significantly increased mRNA levels and density of neurons that express ENK in the hypothalamic paraventricular nucleus and central nucleus of the amygdala, OX, and another orexigenic peptide, melanin-concentrating hormone, in the perifornical lateral hypothalamus in preweanling offspring. These effects persisted in the absence of nicotine, at least until puberty. Colabeling of the cell proliferation marker BrdU with the neuronal marker NeuN and peptides revealed a marked stimulatory effect of prenatal nicotine on neurogenesis, but not gliogenesis, and also on the number of newly generated neurons expressing ENK, OX, or melanin-concentrating hormone. During adolescence, offspring also exhibited significant behavioral changes, increased consumption of nicotine and other substances of abuse, ethanol and a fat-rich diet, with no changes in chow and water intake or body weight. These findings reveal a marked sensitivity during gestation of the orexigenic peptide neurons to low nicotine doses that may increase the offspring's propensity to overconsume substances of abuse during adolescence. PMID:23966683

  9. Asymmetrical myocardial expression of natriuretic peptides in pacing-induced heart failure.

    PubMed

    Del Ry, Silvia; Cabiati, Manuela; Lionetti, Vincenzo; Simioniuc, Anca; Caselli, Chiara; Prescimone, Tommaso; Emdin, Michele; Giannessi, Daniela

    2009-09-01

    High-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive heart failure (HF) with pronounced differences in regional contractility. Aim of this study was to evaluate possible changes in brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) as compared to the infero-septal region (opposite site, OS) and to explore possible association between the contractiling pattern and biomarker expression. Cardiac tissue was collected from minipigs with pacing-induced HF (n=8) and without (control, n=6). The samples were selectively harvested from the anterior left ventricular (LV) wall, PS, and from an area remote to the pacing-site, OS. BNP and CNP mRNA expression was evaluated by semi-quantitative polymerase chain reaction (PCR). A significant difference in BNP expression was found in the PS between HF animals and controls (BNP/GAPDH: 0.65+/-0.11 vs. 0.35+/-0.04, p=0.02), but not in the OS (BNP/GAPDH: 0.36+/-0.05, ns vs. controls). CNP expression was not different compared to controls, although higher levels were observed in the PS and in the OS with respect to the controls (CNP/GAPDH: controls 0.089+/-0.036, PS 0.289+/-0.23, OS 0.54+/-0.16). This finding was in tune with an increase of CNP tissue concentration (controls: 0.69+/-0.13; PS=1.56+/-0.19; OS=1.70+/-0.42 pg/mg protein; p=0.039 controls vs. OS). Higher BNP mRNA expression in the PS is consistent with a reduction in contractile function in this region, while higher CNP mRNA expression in the OS suggests the presence of concomitant endothelial dysfunction in the remote region.

  10. Mucosal Gene Expression of Antimicrobial Peptides in Inflammatory Bowel Disease Before and After First Infliximab Treatment

    PubMed Central

    Lemaire, Katleen; Quintens, Roel; Van Lommel, Leentje; Van Steen, Kristel; Leemans, Peter; Cleynen, Isabelle; Van Assche, Gert; Vermeire, Séverine; Geboes, Karel; Schuit, Frans; Rutgeerts, Paul

    2009-01-01

    Background Antimicrobial peptides (AMPs) protect the host intestinal mucosa against microorganisms. Abnormal expression of defensins was shown in inflammatory bowel disease (IBD), but it is not clear whether this is a primary defect. We investigated the impact of anti-inflammatory therapy with infliximab on the mucosal gene expression of AMPs in IBD. Methodology/Principal Findings Mucosal gene expression of 81 AMPs was assessed in 61 IBD patients before and 4–6 weeks after their first infliximab infusion and in 12 control patients, using Affymetrix arrays. Quantitative real-time reverse-transcription PCR and immunohistochemistry were used to confirm microarray data. The dysregulation of many AMPs in colonic IBD in comparison with control colons was widely restored by infliximab therapy, and only DEFB1 expression remained significantly decreased after therapy in the colonic mucosa of IBD responders to infliximab. In ileal Crohn's disease (CD), expression of two neuropeptides with antimicrobial activity, PYY and CHGB, was significantly decreased before therapy compared to control ileums, and ileal PYY expression remained significantly decreased after therapy in CD responders. Expression of the downregulated AMPs before and after treatment (DEFB1 and PYY) correlated with villin 1 expression, a gut epithelial cell marker, indicating that the decrease is a consequence of epithelial damage. Conclusions/Significance Our study shows that the dysregulation of AMPs in IBD mucosa is the consequence of inflammation, but may be responsible for perpetuation of inflammation due to ineffective clearance of microorganisms. PMID:19956723

  11. Diurnal gene expression of lipolytic natriuretic peptide receptors in white adipose tissue.

    PubMed

    Smith, Julie; Fahrenkrug, Jan; Jørgensen, Henrik L; Christoffersen, Christina; Goetze, Jens P

    2015-12-01

    Disruption of the circadian rhythm can lead to obesity and cardiovascular disease. In white adipose tissue, activation of the natriuretic peptide receptors (NPRs) stimulates lipolysis. We have previously shown that natriuretic peptides are expressed in a circadian manner in the heart, but the temporal expression profile of their cognate receptors has not been examined in white adipose tissue. We therefore collected peri-renal white adipose tissue and serum from WT mice. Tissue mRNA contents of NPRs - NPR-A and NPR-C, the clock genes Per1 and Bmal1, and transcripts involved in lipid metabolism were quantified at 4-h intervals: in the diurnal study, mice were exposed to a period of 12 h light followed by 12 h darkness (n=52). In the circadian study, mice were kept in darkness for 24 h (n=47). Concomitant serum concentrations of free fatty acids, glycerol, triglycerides (TGs), and insulin were measured. Per1 and Bmal1 mRNA contents showed reciprocal circadian profiles (P<0.0001). NPR-A mRNA contents followed a temporal pattern (P=0.01), peaking in the dark (active) period. In contrast, NPR-C mRNA was expressed in an antiphase manner with nadir in the active period (P=0.007). TG concentrations in serum peaked in the active dark period (P=0.003). In conclusion, NPR-A and NPR-C gene expression is associated with the expression of clock genes in white adipose tissue. The reciprocal expression may thus contribute to regulate lipolysis and energy homeostasis in a diurnal manner.

  12. Physiological characterization of formyl peptide receptor expressing cells in the mouse vomeronasal organ.

    PubMed

    Ackels, Tobias; von der Weid, Benoît; Rodriguez, Ivan; Spehr, Marc

    2014-01-01

    The mouse vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Based on largely monogenic expression of either type 1 or 2 vomeronasal receptors (V1Rs/V2Rs) or members of the formyl peptide receptor (FPR) family, the vomeronasal sensory epithelium harbors at least three neuronal subpopulations. While various neurophysiological properties of both V1R- and V2R-expressing neurons have been described using genetically engineered mouse models, the basic biophysical characteristics of the more recently identified FPR-expressing vomeronasal neurons have not been studied. Here, we employ a transgenic mouse strain that coexpresses an enhanced variant of yellow fluorescent protein together with FPR-rs3 allowing to identify and analyze FPR-rs3-expressing neurons in acute VNO tissue slices. Single neuron electrophysiological recordings allow comparative characterization of the biophysical properties inherent to a prototypical member of the FPR-expressing subpopulation of VNO neurons. In this study, we provide an in-depth analysis of both passive and active membrane properties, including detailed characterization of several types of voltage-activated conductances and action potential discharge patterns, in fluorescently labeled vs. unmarked vomeronasal neurons. Our results reveal striking similarities in the basic (electro) physiological architecture of both transgene-expressing and non-expressing neurons, confirming the suitability of this genetically engineered mouse model for future studies addressing more specialized issues in vomeronasal FPR neurobiology. PMID:25484858

  13. Analysis of HLA class I expression in different metastases from two melanoma patients undergoing peptide immunotherapy.

    PubMed

    Mendez, R; Serrano, A; Jäger, E; Maleno, I; Ruiz-Cabello, F; Knuth, A; Garrido, F

    2001-06-01

    We characterized the HLA class I alterations in five metastases obtained from two patients with melanoma immunized with Melan A/MART-1, tyrosinase and gp100 tumor peptides. All three metastases analyzed in the first patient (NW145) showed a similar HLA class I alteration with a dual population of melanoma cells. One population was HLA class I antigen positive and the other had loss of heterozygosity (LOH) in the short arm of chromosome 6 leading to an HLA haplotype loss (A02011, B4007, Cw1). The absence of HLA-A2 antigen may explain why this patient did not develop HLA-A2 restricted, Melan A/MART-1 specificity immunization, since this HLA molecule is the restriction element for the tumor peptides used. However, this HLA-deficient population was not selected after peptide immunotherapy. The primary tumor in this patient presented LOH in region 6q, but only in the vertical growth phase of the lesion, whereas LOH at 6p was observed only in DNA from metastatic material. The second patient (NW16) also presented two metastatic lesions with an identical HLA molecular defect, i.e. HLA B locus downregulation (HLA B51011: serological B51; B1503: serological B70). One lesion expressed the tumor antigen (Melan A/ MART-1), but the other did not. Interestingly, the antigen-positive metastasis regressed after peptide immunotherapy, whereas the other progressed rapidly. These findings provide the first indication that multiple metastases generated in the same host can have identically altered HLA class I phenotypes.

  14. [Four-week simulated weightlessness increases the expression of atrial natriuretic peptide in the myocardium].

    PubMed

    Zhang, Wen-Cheng; Lu, Yuan-Ming; Yang, Huai-Zhang; Xu, Peng-Tao; Chang, Hui; Yu, Zhi-Bin

    2013-04-25

    One of the major circulatory changes that occur in human during space flight and simulated weightlessness is a cerebral redistribution of body fluids, which is accompanied by an increase of blood volume in the upper body. Therefore, atrial myocardium should increase the secretion of atrial natriuretic peptide (ANP), but the researches lack common conclusion until now. The present study was to investigate the expression level of ANP in simulated weightlessness rats, and to confirm the changes of ANP by observing the associated proteins of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The tail-suspended rat model was used to simulate weightlessness. Western blots were carried out to examine the expression levels of ANP and SNARE proteins in atrial and left ventricular myocardium. The results showed that ANP expression in atrial myocardium showed an increase in 4-week tail-suspended rats (SUS) compared with that in the synchronous control rats (CON). We only detected a trace amount of ANP in the left ventricular myocardium of the CON, but found an enhanced expression of ANP in left ventricular myocardium of the SUS. Expression of VAMP-1/2 (vesicle associated SNARE) increased significantly in both atrial and left ventricular myocardium in the SUS compared with that in the CON. There was no difference of the expression of syntaxin-4 (target compartment associated SNARE) between the CON and SUS, but the expression of SNAP-23 showed an increase in atrial myocardium of the SUS compared with that in the CON. Synip and Munc-18c as regulators of SNAREs did not show significant difference between the CON and SUS. These results suggest that the expression of ANP shows an increase in atrial and left ventricular myocardium of 4-week tail-suspended rats. Enhanced expression of VAMP-1/2 associated with ANP vesicles confirms the increased expression of ANP in atrial and left ventricular myocardium.

  15. Expression and potential role of the peptide orexin-A in prostate cancer

    SciTech Connect

    Valiante, Salvatore; Liguori, Giovanna; Tafuri, Simona; Pavone, Luigi Michele; Campese, Roberto; Monaco, Roberto; Iachetta, Giuseppina; Assisi, Loredana; Mirabella, Nicola; Forte, Maurizio; Costagliola, Anna; Vittoria, Alfredo

    2015-09-04

    The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A in human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer. - Highlights: • Orexin-A and OX1 receptor are present in human cancer prostate tissues. • Orexin-A up-regulates OX1 receptor expression in LNCaP cells. • Orexin-A inhibits testosterone-induced nuclear translocation of androgen receptor.

  16. Expression profiles of antimicrobial peptides (AMPs) and their regulation by Relish

    NASA Astrophysics Data System (ADS)

    Wang, Dongdong; Li, Fuhua; Li, Shihao; Wen, Rong; Xiang, Jianhai

    2012-07-01

    Antimicrobial peptides (AMPs), as key immune effectors, play important roles in the innate immune system of invertebrates. Different types of AMPs, including Penaeidin, Crustin, ALF (antilipopolysaccharide factor) have been identified in different penaeid shrimp; however, systematic analyses on the function of different AMPs in shrimp responsive to different types of bacteria are very limited. In this study, we analyzed the expression profiles of AMPs in the Chinese shrimps, Fenneropenaeus chinensis, simultaneously by real-time RT-PCR (reverse transcription-polymerase chain reaction) when shrimp were challenged with Micrococcus lysodeikticus (Gram-positive, G+) or Vibrio anguillarium (Gram-negative, G-). Different AMPs showed different expression profiles when shrimp were injected with one type of bacterium, and one AMP also showed different expression profiles when shrimp were challenged with different bacteria. Furthermore, the expression of these AMPs showed temporal expression profiles, suggesting that different AMPs function coordinately in bacteria-infected shrimp. An RNA interference approach was used to study the function of the Relish transcription factor in regulating the transcription of different AMPs. The current study showed that Relish could regulate the transcription of different AMPs in shrimp. Differential expression profiles of AMPs in shrimp injected with different types of bacteria indicated that a complicated antimicrobial response network existed in shrimp. These data contribute to our understanding of immunity in shrimp and may provide a strategy for the control of disease in shrimp.

  17. Involvement of Relish gene from Macrobrachium rosenbergii in the expression of anti-microbial peptides.

    PubMed

    Shi, Yan-Ru; Jin, Min; Ma, Fu-Tong; Huang, Ying; Huang, Xin; Feng, Jin-Ling; Zhao, Ling-Ling; Chen, Yi-Hong; Ren, Qian

    2015-10-01

    Relish is an NF-kB transcription factor involved in immune-deficiency (IMD) signal pathway. In this study, a Relish gene (MrRelish) was identified from Macrobrachium rosenbergii. The full length of MrRelish comprises 5072 bp, including a 3510 bp open reading frame encoding a 1169 bp amino acid protein. MrRelish contains a Rel homology domain (RHD), a nucleus localization signal, an IκB-like domain (6 ankyrin repeats), and a death domain. Phylogenetic analysis showed that MrRelish and other Relish from crustaceans belong to one group. MrRelish was expressed in all detected tissues, with the highest expression level in hemocytes and intestines. MrRelish was also upregulated in hepatopancreas at 6 h after Vibrio anguillarum challenge. The over-expression of MrRelish could induce the expression of antimicrobial peptides (AMPs), such as Drosophila Metchnikowin (Mtk), Attacin (Atta), Drosomycin (Drs), and Cecropin (CecA) and shrimp Penaeidin (Pen4). The RNAi of MrRelish in gills showed that the expression of crustin (cru) 2, Cru5, Cru8, lysozyme (Lyso) 1, and Lyso2 was inhibited. However, the expression of anti-lipopolysaccharide factor (ALF) 1 and ALF3 did not change when MrRelish was knocked down. These results indicate that MrRelish may play an important role in innate immune defense against V. anguillarum in M. rosenbergii.

  18. Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif.

    PubMed

    Riahi, Nesrine; Cappadocia, Laurent; Henry, Olivier; Omichinski, James; De Crescenzo, Gregory

    2016-02-01

    A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides. PMID:26459292

  19. The Melanocortin-4 Receptor is Expressed in Enteroendocrine L Cells and Regulates the Release of Peptide YY and Glucagon-Like Peptide 1 In Vivo

    PubMed Central

    Panaro, Brandon L.; Tough, Iain R.; Engelstoft, Maja Storm; Matthews, Robert T.; Digby, Gregory J.; Møller, Cathrine Laustrup; Svendsen, Berit; Gribble, Fiona; Reimann, Frank; Holst, Jens J.; Holst, Birgitte; Schwartz, Thue W.; Cox, Helen M.; Cone, Roger D.

    2014-01-01

    SUMMARY The melanocortin-4 receptor (MC4R) is expressed in the brainstem and vagal afferent nerves, and regulates a number of aspects of gastrointestinal function. Here we show that the receptor is also diffusely expressed in cells of the gastrointestinal system, from stomach to descending colon. Furthermore, MC4R is the second most highly expressed GPCR in peptide YY (PYY) and glucagon-like peptide one (GLP-1) expressing enteroendocrine L cells. When vectorial ion transport is measured across mouse or human intestinal mucosa, administration of α-MSH induces a MC4R-specific PYY-dependent anti-secretory response consistent with a role for the MC4R in paracrine inhibition of electrolyte secretion. Finally, MC4R-dependent acute PYY and GLP-1 release from L cells can be stimulated in vivo by intraperitoneal administration of melanocortin peptides to mice. This suggests physiological significance for MC4R in L cells, and indicates a previously unrecognized peripheral role for the MC4R, complementing vagal and central receptor functions. PMID:25453189

  20. Gastrin-releasing peptide expression and its effect on the calcification of developing mouse incisor.

    PubMed

    Lee, Dong-Joon; Jin, Chengri; Kim, Eun-Jung; Lee, Jong-Min; Jung, Han-Sung

    2015-09-01

    Gastrin-releasing peptide (GRP) is considered to be one of the cancer growth factors. This peptide's receptor (GRPR) is known as a G protein-coupled receptor, regulating intracellular calcium storage and releasing signals. This study is the first to investigate the function of GRP during mouse incisor development. We hypothesized that GRP is one of the factors that affects the regulation of calcification during tooth development. To verify the expression pattern of GRP, in situ hybridization was processed during incisor development. GRP was expressed at the late bell stage and hard tissue formation stage in the epithelial tissue. To identify the genuine function of GRP during incisor development, a gain-of-function analysis was performed. After GRP overexpression in culture, the phenotype of ameloblasts, odontoblasts and predentin was altered compared to control group. Moreover, enamel and dentin thickness was increased after renal capsule transplantation of GRP-overexpressed incisors. With these results, we suggest that GRP plays a significant role in the formation of enamel and dentin by regulating ameloblasts and predentin formation, respectively. Thus, GRP signaling is strongly related to calcium acquisition and secretion during mouse incisor development.

  1. Expression and purification of mouse peptide ESP4 in Escherichia coli.

    PubMed

    Hirakane, Makoto; Taniguchi, Masahiro; Yoshinaga, Sosuke; Misumi, Shogo; Terasawa, Hiroaki

    2014-04-01

    Pheromones are species-specific chemical signals that regulate a wide range of social and sexual behaviors in many animals. In mice, the male-specific peptide ESP1 (exocrine gland-secreting peptide 1) is secreted into tear fluids and enhances female sexual receptive behavior. ESP1 belongs to the ESP family, a multigene family with 38 genes in mice. ESP1 shares the highest homology with ESP4. ESP1 is expressed in the extraorbital lacrimal gland, whereas ESP4 is expressed in some exocrine glands. Thus, ESP4 is expected to have a function that has not been elucidated yet. Large amounts of the purified ESP4 protein are required for structural and biochemical studies. Here we present an expression and purification scheme for the recombinant ESP4 protein. The N-terminally histidine-tagged ESP4 fusion protein was expressed in Escherichia coli as inclusion bodies, which were solubilized and purified by nickel affinity chromatography. The histidine tag was cleaved with thrombin and removed by a second nickel affinity chromatography step. The ESP4 protein was isolated with high purity by reversed-phase chromatography. For NMR analyses, we prepared a stable isotope-labeled ESP4 protein. Three repeated freeze-drying steps after the reversed-phase chromatography were required, to remove a volatile contaminating compound and to obtain an NMR spectrum with a homogeneous line shape. AMS-modification and far-UV CD spectroscopic analyses suggested that ESP4 has an intramolecular disulfide bridge and a helical structure, respectively. The present study provides a powerful tool for structural and biochemical studies of ESP4, leading toward the elucidation of the roles of the ESP family members.

  2. Molecular cloning, expression analysis, and appetite regulatory effect of peptide YY in Siberian sturgeon (Acipenser baerii).

    PubMed

    Chen, Hu; Zhang, Xin; Hao, Jin; Chen, Defang; Liu, Ju; Gao, Yundi; Zhu, Jieyao; Wu, Hongwei; Lin, Fangjun; Pu, Yundan; Yuan, Dengyue; Wei, Rongbin; Zhou, Chaowei; Wang, Tao; Li, Zhiqiong

    2015-06-01

    Peptide YY (PYY) is an anorectic brain-gut peptide involved in feeding regulation and well characterized in mammals. However, the functional role of PYY in the appetite regulatory of fish is not clear. In this study, we characterized a high conservation of PYY cDNA and found high expression levels of PYY mRNA in the brain and digestive tract of Siberian sturgeon. Then, we examined preprandial (pre- and post-feeding) changes of PYY mRNA expression in the brain that showed a significantly increased in 3h post-feeding, suggesting an anorectic possible function of PYY in Siberian sturgeon. Next, we examined the expression of PYY mRNA during 15 days fasting and refeed after fasting. The SsPYY mRNA expression of unfed fish had a significant 2.4, 1.7, 2.0, 2.2, and 2.1-fold decrease compared to 1-, 3-, 6-, 10- and 15-day ad libitum fed animals, respectively. After refeed, SsPYY mRNA significantly increased 1.9 and 4.1-fold above that of the 15-day fed and unfed fish control group (P<0.01). Furthermore, a single intraperitoneal injection of 10, 100 and 200 ng/g BW SsPYY1-36 caused a reduction in the next feeding and no significant reduction in food intake was observed in fish injected with a 1 ng/g BW. Overall, PYY has a potentially role in food intake attenuation of Siberian sturgeon.

  3. Production of antibacterial peptide from bee venom via a new strategy for heterologous expression.

    PubMed

    Hou, Chunsheng; Guo, Liqiong; Lin, Junfang; You, Linfeng; Wu, Wuhua

    2014-12-01

    Honey bee is important economic insect that not only pollinates fruits and crops but also provides products with various physiological activities. Bee venom is a functional agent that is widely applied in clinical treatment and pharmacy. Secapin is one of these agents that have a significant role in therapy. The functions of secapin from the bee venom have been documented, but little information is known about its heterologous expression under natural condition. Moreover, few scholars verified experimentally the functions of secapin from bee venom in vitro. In this study, we successfully constructed a heterologous expression vector, which is different from conventional expression system. A transgenic approach was established for transformation of secapin gene from the venom of Apis mellifera carnica (Ac-sec) into the edible fungi, Coprinus cinereus. Ac-sec was encoded by a 234 bp nucleotide that contained a signal peptide domain and two potential phosphorylation sites. The sequence exhibited highly homology with various secapins characterized from honey bee and related species. Southern blot data indicated that Ac-sec was present as single or multiple copy loci in the C. cinereus genome. By co-transformation and double-layer active assay, Ac-sec was expressed successfully in C. cinereus and the antibacterial activity of the recombinants was identified, showing notable antibacterial activities on different bacteria. Although Ac-sec is from the venom of Apidae, phylogenetic analysis demonstrated that Ac-sec was more closely related to that of Vespid than to bee species from Apidae. The molecular characteristics of Ac-sec and the potential roles of small peptides in biology were discussed. PMID:25189650

  4. Gene expression for peptides in neurons of the petrosal and nodose ganglia in rat.

    PubMed

    Czyzyk-Krzeska, M F; Bayliss, D A; Seroogy, K B; Millhorn, D E

    1991-01-01

    In situ hybridization was used to determine whether genes for neuropeptides [substance P/neurokinin A (SP/NKA), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide tyrosine (NPY) and cholecystokinin (CCK)] are expressed in inferior ganglia of the vagus (nodose) and glossopharyngeal (petrosal) nerves. Synthetic oligodeoxyribonucleotides, complementary to the cognate, mRNAs were labeled with [32P] or [35S], and hybridized to 10 microns thick sections of unperfused tissue which were then processed for film and emulsion autoradiography. We found numerous, clustered neuronal perikarya throughout the nodose and petrosal ganglia that expressed preprotachykinin A (SP/NKA) and CGRP mRNAs to varying degrees. Neurons expressing preproSOM mRNA were less abundant and more scattered throughout both ganglia. Notably, we found mRNA for NPY in cells (usually 5-10 per section) in both ganglia. To our knowledge, this is first evidence for NPY in these sensory ganglia. In contrast to previous immunohistochemical findings, we found no evidence for expression of preproCCK in either the nodose or petrosal ganglia. The present findings demonstrate that cells of the nodose and petrosal ganglia express the genes for a number of neuropeptides that are presumably involved with transmission of visceral sensory afferent information to higher order neurons of the central nervous system. PMID:1708726

  5. Estradiol-induced hypophagia is associated with the differential mRNA expression of hypothalamic neuropeptides.

    PubMed

    Silva, L E C M; Castro, M; Amaral, F C; Antunes-Rodrigues, J; Elias, L L K

    2010-08-01

    Estradiol participates in the control of energy homeostasis, as demonstrated by an increase in food intake and in body weight gain after ovariectomy in rats. In the present study, female Wistar rats (200-230 g, N = 5-15 per group), with free access to chow, were individually housed in metabolic cages. We investigated food intake, body weight, plasma leptin levels, measured by specific radioimmunoassay, and the hypothalamic mRNA expression of orexigenic and anorexigenic neuropeptides, determined by real-time PCR, in ovariectomized rats with (OVX+E) and without (OVX) estradiol cypionate treatment (10 microg/kg body weight, sc, for 8 days). Hormonal and mRNA expression were determined at pre-feeding and 4 h after food intake. OVX+E rats showed lower food intake, less body weight gain and lower plasma leptin levels. In the OVX+E group, we also observed a reduction of neuropeptide Y (NPY), agouti-related protein (AgRP) and cocaine- and amphetamine-regulated transcript (CART) mRNA expression in the arcuate nucleus and a decrease in orexin A in the lateral hypothalamic area (LHA). There was an increase in leptin receptor (LepRb), melanocortin-4 receptor (MC4-R), CART, and mainly corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus and LepRb and CART mRNA in the LHA. These data show that hypophagia induced by estradiol treatment is associated with reduced hypothalamic expression of orexigenic peptides such as NPY, AgRP and orexin A, and increased expression of the anorexigenic mediators MC4-R, LepRb and CRH. In conclusion, estradiol decreases food intake, and this effect seems to be mediated by peripheral factors such as leptin and the differential mRNA expression of neuropeptides in the hypothalamus.

  6. The putative signal peptide of glucagon-like peptide-1 receptor is not required for receptor synthesis but promotes receptor expression

    PubMed Central

    Ge, Yunjun; Yang, Dehua; Dai, Antao; Zhou, Caihong; Zhu, Yue; Wang, Ming-Wei

    2014-01-01

    GLP-1R (glucagon-like peptide-1 receptor) mediates the ‘incretin effect’ and many other anti-diabetic actions of its cognate ligand, GLP-1 (glucagon-like peptide-1). It belongs to the class B family of GPCRs (G protein-coupled receptors) and possesses an N-terminal putative SP (signal peptide). It has been reported that this sequence is required for the synthesis of GLP-1R and is cleaved after receptor synthesis. In the present study, we conducted an in-depth exploration towards the role of the putative SP in GLP-1R synthesis. A mutant GLP-1R without this sequence was expressed in HEK293 cells (human embryonic kidney 293 cells) and displayed normal functionality with respect to ligand binding and activation of adenylate cyclase. Thus the putative SP does not seem to be required for receptor synthesis. Immunoblotting analysis shows that the amount of GLP-1R synthesized in HEK293 cells is low when the putative SP is absent. This indicates that the role of the sequence is to promote the expression of GLP-1R. Furthermore, epitopes tagged at the N-terminal of GLP-1R are detectable by immunofluorescence and immunoblotting in our experiments. In conclusion, the present study points to different roles of SP in GLP-1R expression which broadens our understanding of the functionality of this putative SP of GLP-1R and possibly other Class B GPCRs. PMID:25330813

  7. Enzyme immunoassay detection of induction of MHC class I expression by synthetic peptides from the E6 and E7 regions of human papillomavirus type 16.

    PubMed

    Dillner, J

    1994-01-01

    Viral antigens are presented to cytotoxic T cells (CTL) in the form of endogenously processed peptides bound to major histocompatibility complex (MHC) class I molecules. A variety of different methods for measuring the ability of peptides to bind to MHC class I have been described. Several of these methods use the murine lymphoma mutant cell line RMA-S, which has a peptide loading defect resulting in a low expression of surface class I molecules that can be upregulated if a synthetic binding peptide with class I binding ability is added to the culture medium. In order to be able to screen for peptides with MHC class I binding ability, we developed an enzyme immunoassay for quantitation of MHC class I expression on RMA-S cells. 107 synthetic peptides derived from the E6 and E7 regions of human papillomavirus type 16 were screened for ability to upregulate class I expression of Kb or Db alleles. At a concentration of about 300 microM, 9/107 peptides were found to restore expression of Db to equal or greater levels than found in the RMA-S parental cell line RMA, while 35/107 peptides were able to partially restore Db expression. For Kb, 16/107 peptides were able to restore expression and 40/107 peptides induced partial upregulation. Titration experiments showed that upregulation of class I expression by these peptides was dependent on a high peptide concentration, since consistent upregulation could in no case be detected at concentrations below 10 microM. The class I binding peptides identified in the present study may be useful in the study of the CTL response to HPV in mouse model systems. The enzyme immunoassay used could facilitate the rapid search for class I binding peptides.

  8. Expression of essential genes for biosynthesis of antimicrobial peptides of Bacillus is modulated by inactivated cells of target microorganisms.

    PubMed

    Leães, Fernanda Leal; Velho, Renata Voltolini; Caldas, Danielle Gregório Gomes; Ritter, Ana Carolina; Tsai, Siu Mui; Brandelli, Adriano

    2016-01-01

    Certain Bacillus strains are important producers of antimicrobial peptides with great potential for biological control. Antimicrobial peptide production by Bacillus amyloliquefaciens P11 was investigated in the presence of heat-inactivated cells of bacteria and fungi. B. amyloliquefaciens P11 exhibited higher antimicrobial activity in the presence of inactivated cells of Staphylococcus aureus and Aspergillus parasiticus compared to other conditions tested. Expression of essential genes related to biosynthesis of the antimicrobial peptides surfactin (sfp), iturin A (lpa-14 and ituD), subtilosin A (sboA) and fengycin (fenA) was investigated by quantitative real-time PCR (qRT-PCR). The genes lpa-14 and ituD were highly expressed in the presence of S. aureus (inactivated cells), indicating induction of iturin A production by B. amyloliquefaciens P11. The other inducing condition (inactivated cells of A. parasiticus) suppressed expression of lpa-14, but increased expression of ituD. A twofold increase in fenA expression was observed for both conditions, while strong suppression of sboA expression was observed in the presence of inactivated cells of S. aureus. An increase in antimicrobial activity was observed, indicating that synthesis of antimicrobial peptides may be induced by target microorganisms. PMID:26577655

  9. The effect of peptidoglycan enriched diets on antimicrobial peptide gene expression in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Casadei, Elisa; Bird, Steve; Vecino, Jose L González; Wadsworth, Simon; Secombes, Christopher J

    2013-02-01

    The aim of this study was to investigate the effect of feeding rainbow trout (Oncorhynchus mykiss) peptidoglycan (PG) enriched diets on antimicrobial peptide (AMP) gene expression. Fish were divided into 5 groups and fed diets containing 0, 5, 10, 50 and 100 mg PG/Kg, and sampled 1, 7 and 14 days later. The expression of eight AMP genes (four defensins, two cathelicidins and two liver expressed AMPs) was determined in skin, gill, gut and liver, tissues important for first lines of defence or production of acute phase proteins. Up-regulation of many AMPs was found after feeding the PG enriched diets, with sequential expression seen over the time course studied, where defensins were typically expressed early and cathelicidins and LEAPs later on. A number of clear differences in AMP responsiveness between the tissues examined were also apparent. Of the four PG concentrations used, 5 mg PG/Kg did not always elicit AMP gene induction or to the same degree as seen with the other diets. The three higher dose groups generally showed similar trends although differences in fold change were more pronounced in the 50 and 100 mg PG/Kg groups. Curiously several AMPs were down-regulated after 14 days of feeding in gills, gut and liver. Nevertheless, overall the PG enriched diets had a positive effect on AMP expression. Further investigations now need to be undertaken to confirm whether this higher AMP gene expression correlates with protection against common bacterial diseases and if PG enriched diets have value as a means to temporarily boost the piscine immune system. PMID:23220715

  10. Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression.

    PubMed

    Wang, G; Xu, X; Pace, B; Dean, D A; Glazer, P M; Chan, P; Goodman, S R; Shokolenko, I

    1999-07-01

    Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases.

  11. Isotretinoin therapy changes the expression of antimicrobial peptides in acne vulgaris.

    PubMed

    Borovaya, Alena; Dombrowski, Yvonne; Zwicker, Stephanie; Olisova, Olga; Ruzicka, Thomas; Wolf, Ronald; Schauber, Jürgen; Sárdy, Miklós

    2014-10-01

    In acne vulgaris, antimicrobial peptides (AMPs) could play a dual role; i.e., protective by acting against Propionibacterium acnes, pro-inflammatory by acting as signalling molecules. The cutaneous expression of 15 different AMPs was investigated in acne patients; furthermore, the impact of isotretinoin therapy on AMP expression was analysed in skin biopsies from 13 patients with acne vulgaris taken before, during and after a 6-month treatment cycle with isotretinoin using quantitative real-time polymerase chain reaction. Cutaneous expression of the AMPs cathelicidin, human β-defensin-2 (HBD-2), lactoferrin, lysozyme, psoriasin (S100A7), koebnerisin (S100A15), and RNase 7 was upregulated in untreated acne vulgaris, whereas α-defensin-1 (HNP-1) was downregulated compared to controls. While relative expression levels of cathelicidin, HBD-2, lactoferrin, psoriasin (S100A7), and koebnerisin (S100A15) decreased during isotretinoin treatment, only those of cathelicidin and koebnerisin returned to normal after 6 months of isotretinoin therapy. The increased expression of lysozyme and RNase 7 remained unaffected by isotretinoin treatment. The levels of granulysin, RANTES (CCL5), perforin, CXCL9, substance P, chromogranin B, and dermcidin were not regulated in untreated acne patients and isotretinoin had no effect on these AMPs. In conclusion, the expression of various AMPs is altered in acne vulgaris. Isotretinoin therapy normalizes the cutaneous production of distinct AMPs while the expression of others is still increased in healing acne. Considering the antimicrobial and pro-inflammatory role of AMPs, these molecules could serve as specific targets for acne therapy and maintenance of clinical remission.

  12. Expression, purification, and C-terminal amidation of recombinant human glucagon-like peptide-1.

    PubMed

    Zhang, Zhi-Zhen; Yang, Sheng-Sheng; Dou, Hong; Mao, Ji-Fang; Li, Kang-Sheng

    2004-08-01

    Human glucagon-like peptide-1 (hGLP-1) (7-36) amide, a gastrointestinal hormone with a pharmaceutical potential in treating type 2 diabetes mellitus, is composed of 30 amino acid residues as a mature protein. We report here the development of a method for high-level expression and purification of recombinant hGLP-1 (7-36) amide (rhGLP-1) through glutathione S-transferase (GST) fusion expression system. The cDNA of hGLP-1-Leu, the 31st-residue leucine-extended precursor peptide, was prepared by annealing and ligating of artificially synthetic oligonucleotide fragments, inserted into pBluescript SK (+/-) plasmid, and then cloned into pGEX-4T-3 GST fusion vector. The fusion protein GST-hGLP-1-Leu, expressed in Escherichia coli strain BL21 (DE3), was purified by affinity chromatography after high-level culture and sonication of bacteria. Following cleavage of GST-hGLP-1-Leu by cyanogen bromide, the recombinant hGLP-1-Leu was released from fusion protein, and purified using QAE Sepharose ion exchange and RP C(18) chromatography. After purification, the precursor hGLP-1-Leu was transacylated by carboxypeptidase Y, Arg-NH(2) as a nucleophile, to produce rhGLP-1. Electrospray ionization mass spectrometry showed the molecular weight was as expected. The biological activity of rhGLP-1 in a rat model demonstrated that plasma glucose concentrations were significantly lower and insulin concentrations higher after intraperitoneal injection of rhGLP-1 together with glucose compared with glucose alone (P < 0.001). PMID:15249052

  13. CART peptide and opioid addiction: Expression changes in male rat brain.

    PubMed

    Bakhtazad, A; Vousooghi, N; Garmabi, B; Zarrindast, M R

    2016-06-14

    Previous studies have shown the prominence of cocaine- and amphetamine-regulated transcript (CART) peptide in rewarding and reinforcing effects of drugs of abuse specially psychostimulants. The data regarding the effects of different stages of opioid addiction on CART expression and the interconnection between CART and opioids are not much available. Here we have studied the changes in the expression level of CART mRNA and protein in various parts of the brain reward pathway in different stages of opioid addiction. Groups of male rats received acute low-dose (10mg/kg), acute high-dose (80mg/kg) and chronic escalating doses of morphine. In addition, withdrawal and abstinence states were evaluated after injection of naloxone (1mg/kg) and long-term maintenance of addicted animals, respectively. Expression of CART mRNA in the brain was measured by real-time PCR method. Western blotting was used to quantify the protein level. CART mRNA and protein were both up-regulated in high-dose morphine-administered animals and also in the withdrawal group in the nucleus accumbens (NAc), striatum and prefrontal cortex (PFC). In the addicted group, CART mRNA and protein were both down-regulated in NAc and striatum. In the abstinent group, CART mRNA was down-regulated in NAc. In the hippocampus, the only observed change was the up-regulation of CART mRNA in the withdrawal group. We suggest that the modulatory role of CART peptide in rewarding and reinforcing effects of opioids weakens when opioids are used for a long time and is stimulated when acute stress such as naloxone-induced withdrawal syndrome or acute high-dose administration of morphine occurs to the animal.

  14. Expressed Peptide Tags: An additional layer of data for genome annotation

    SciTech Connect

    Savidor, Alon; Donahoo, Ryan S; Hurtado-Gonzales, Oscar; Verberkmoes, Nathan C; Shah, Manesh B; Lamour, Kurt H; McDonald, W Hayes

    2006-01-01

    While genome sequencing is becoming ever more routine, genome annotation remains a challenging process. Identification of the coding sequences within the genomic milieu presents a tremendous challenge, especially for eukaryotes with their complex gene architectures. Here we present a method to assist the annotation process through the use of proteomic data and bioinformatics. Mass spectra of digested protein preparations of the organism of interest were acquired and searched against a protein database created by a six frame translation of the genome. The identified peptides were mapped back to the genome, compared to the current annotation, and then categorized as supporting or extending the current genome annotation. We named the classified peptides Expressed Peptide Tags (EPTs). The well annotated bacterium Rhodopseudomonas palustris was used as a control for the method and showed high degree of correlation between EPT mapping and the current annotation, with 86% of the EPTs confirming existing gene calls and less than 1% of the EPTs expanding on the current annotation. The eukaryotic plant pathogens Phytophthora ramorum and Phytophthora sojae, whose genomes have been recently sequenced and are much less well annotated, were also subjected to this method. A series of algorithmic steps were taken to increase the confidence of EPT identification for these organisms, including generation of smaller sub-databases to be searched against, and definition of EPT criteria that accommodates the more complex eukaryotic gene architecture. As expected, the analysis of the Phytophthora species showed less correlation between EPT mapping and their current annotation. While ~77% of Phytophthora EPTs supported the current annotation, a portion of them (7.2% and 12.6% for P. ramorum and P. sojae, respectively) suggested modification to current gene calls or identified novel genes that were missed by the current genome annotation of these organisms.

  15. Strategies for bacterial expression of protein-peptide complexes: application to solubilization of Papillomavirus E6

    PubMed Central

    Sidi, Abdellahi Ould M’hamed Ould; Babah, Khaled Ould; Brimer, Nicole; Nominé, Yves; Romier, Christophe; Kieffer, Bruno; Pol, Scott Vande; Travé, Gilles; Zanier, Katia

    2011-01-01

    E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognises its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that Bovine Papillomavirus Type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein’s solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein MBP (Maltose-Binding-Protein), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif. PMID:21777678

  16. Cholecalciferol (vitamin D) differentially regulates antimicrobial peptide expression in bovine mammary epithelial cells: implications during Staphylococcus aureus internalization.

    PubMed

    Téllez-Pérez, Ana Dolores; Alva-Murillo, Nayeli; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

    2012-11-01

    Vitamin D has immunomodulatory functions regulating the expression of host defense genes. The aim of this study was to determine the effect of cholecalciferol (vitamin D3) on S. aureus internalization into bovine mammary epithelial cells (bMEC) and antimicrobial peptide (AP) mRNA expression. Cholecalciferol (1-200 nM) did not affect S. aureus growth and bMEC viability; but it reduced bacterial internalization into bMEC (15-74%). Also, bMEC showed a basal expression of all AP genes evaluated, which were induced by S. aureus. Cholecalciferol alone or together with bacteria diminished tracheal antimicrobial peptide (TAP) and bovine neutrophil β-defensin (BNBD) 5 mRNA expression; while alone induced the expression of lingual antimicrobial peptide (LAP), bovine β-defensin 1 (DEFB1) and bovine psoriasin (S100A7), which was inhibited in the presence of S. aureus. This compound (50 nM) increased BNBD10 mRNA expression coinciding with the greatest reduction in S. aureus internalization. Genes of vitamin D pathway (25-hydroxylase and 1 α-hydroxylase) show basal expression, which was induced by cholecalciferol or bacteria. S. aureus induced vitamin D receptor (VDR) mRNA expression, but not in the presence of cholecalciferol. In conclusion, cholecalciferol can reduce S. aureus internalization and differentially regulates AP expression in bMEC. Thus, vitamin D could be an effective innate immunity modulator in mammary gland, which leads to a better defense against bacterial infection.

  17. Orexigenic effects of endomorphin-2 (EM-2) related to decreased CRH gene expression and increased dopamine and norepinephrine activity in the hypothalamus.

    PubMed

    Brunetti, Luigi; Ferrante, Claudio; Orlando, Giustino; Recinella, Lucia; Leone, Sheila; Chiavaroli, Annalisa; Di Nisio, Chiara; Shohreh, Rugia; Manippa, Fabio; Ricciuti, Adriana; Mollica, Adriano; Vacca, Michele

    2013-10-01

    Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are opioid peptides which are selective partial agonists of μ-opioid receptor. We studied the effects of EM-2 injected into the arcuate nucleus (ARC) of the hypothalamus on feeding behavior and gene expression of orexigenic [agouti-related peptide (AgRP), neuropeptide Y (NPY) and orexin-A] and anorexigenic [cocaine and amphetamine-regulated transcript (CART), corticotrophin releasing hormone (CRH) and proopiomelanocortin (POMC)] peptides in male Wistar rats fed a standard laboratory diet. Furthermore, we evaluated the effects of EM-2 on dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) steady state concentrations, in the hypothalamus. 64 rats (16 for each group of treatment) were injected into the ARC, at 9.00 am, with either vehicle or EM-2 (0.50-0.75 μmol/kg) or EM-2 (0.50 μmol/kg) plus β-funaltrexamine (0.20 μmol/kg). Food intake was recorded through 24h following injection, and hypothalamic DA, NE, 5-HT levels and neuropeptide gene expression were evaluated 24h after EM-2 administration. Compared to vehicle, EM-2 significantly increased food intake, throughout 24h post-injection. Furthermore, EM-2 treatment led to a significant increase of DA and NE concentrations and a decrease of CRH mRNA levels. On the other hand, β-funaltrexamine administration reverted both feeding stimulatory and neuromodulatory effects induced by EM-2. We can conclude that the orexigenic effect of μ-opioid receptor activation by EM-2 could be related to both inhibition of CRH and stimulation of dopamine and norepinephrine levels, in the hypothalamus.

  18. Use of Peptide Nucleic Acids to Manipulate Gene Expression in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Naik, Shankar; Yavin, Eylon; Dzikowski, Ron

    2014-01-01

    One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents. PMID:24466246

  19. Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection.

    PubMed

    Pridgeon, J W; Mu, X; Klesius, P H

    2012-03-01

    Using quantitative polymerase chain reaction (QPCR), the relative transcriptional levels of seven channel catfish antimicrobial peptide (AMP) genes (NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability-increasing protein, cathepsin D, hepcidin and liver-expressed AMP 2) in response to Edwardsiella ictaluri infection were determined. None of the AMP genes tested was significantly upregulated at 2 h post-infection. Hepcidin was the only one that was significantly (P<0.05) upregulated at 4, 6 and 12 h post-infection. At 24 and 48 h post-infection, four AMPs (hepcidin, NK-lysin type 1, NK-lysin type 3 and cathepsin D) were significantly (P<0.05) upregulated. Among all the AMPs that were significantly upregulated at different time points, hepcidin at 4, 6 and 12 h post-infection was upregulated the most. When catfish were injected with different doses of E. ictaluri, all lethal doses were able to induce significant (P <0.05) upregulation of hepcidin in the posterior kidney, whereas sublethal doses failed to induce any significant upregulation of hepcidin. In vitro growth studies revealed that the presence of synthetic hepcidin peptide at a concentration of 16 μm or higher significantly inhibited the cell proliferation of E. ictaluri. Taken together, our results suggest that hepcidin might play an important role in the channel catfish defence against E. ictaluri infection.

  20. HYPERPHAGIA INDUCED BY SUCROSE: RELATION TO CIRCULATING AND CSF GLUCOSE AND CORTICOSTERONE AND OREXIGENIC PEPTIDES IN THE ARCUATE NUCLEUS

    PubMed Central

    Gaysinskaya, V. A.; Karatayev, O.; Shuluk, J.; Leibowitz, S. F.

    2010-01-01

    Sucrose-rich diets compared to starch-rich diets are known to stimulate overeating under chronic conditions. The present study in normal-weight rats established an acute “preload-to-test meal” paradigm for demonstrating sucrose-induced hyperphagia and investigating possible mechanisms that mediate this behavioral phenomenon. In this acute paradigm, the rats were first given a small (15 kcals) sucrose preload (30% sucrose) for 30 min compared to an equicaloric, starch preload (25% starch with 5% sucrose) and then allowed to freely consume a subsequent test meal of lab chow. The sucrose preload, when compared to a starch preload equal in energy density and palatability, consistently increased food intake in the subsequent test meal occurring between 60–120 min after the end of the preload. Measurements of hormones, metabolites and hypothalamic peptides immediately preceding this hyperphagia revealed marked differences between the sucrose vs starch groups that could contribute to the increase in food intake. Whereas the sucrose group compared to starch group immediately after the preload (at 10 min) had elevated levels of glucose in serum and cerebrospinal fluid (CSF) along with reduced expression of neuropeptide Y (NPY) and agouti-related protein (AgRP) in the arcuate nucleus (ARC), the subsequent effects (at 30–60 min) just preceding the test meal hyperphagia were the reverse. Along with lower levels of glucose, they included markedly elevated serum and CSF levels of corticosterone and mRNA levels of NPY and AgRP in the ARC. In addition to establishing an animal model for sucrose-induced hyperphagia, these results demonstrate peripheral and central mechanisms that may mediate this behavioral phenomenon. PMID:21036188

  1. Fed and fasted chicks from lines divergently selected for low or high body weight have differential hypothalamic appetite-associated factor mRNA expression profiles.

    PubMed

    Yi, Jiaqing; Gilbert, Elizabeth R; Siegel, Paul B; Cline, Mark A

    2015-06-01

    We have demonstrated that chicken lines which have undergone intense divergent selection for either low (LWS) or high (HWS) body weight (anorexic and obese containing, respectively) have differential food intake threshold responses to a range of intracerebroventricular injected neurotransmitters. The study reported herein was designed to measure endogenous appetite-associated factor mRNA profiles between these lines in an effort to further understand the molecular mechanisms involved in their differential eating patterns. Whole hypothalamus was collected from 5 day-old chicks that had been fasted for 180 min or had free access to food. Total RNA was isolated, reverse transcribed, and real-time PCR performed. Although mRNAs encoding orexigenic neuropeptides including agouti-related peptide, neuropeptide Y (NPY), prolactin-releasing peptide, and visfatin did not differ in expression between the lines, NPY receptor 5 mRNA was greater in fed LWS than HWS chicks, but fasting decreased the magnitude of difference. Anorexigenic factors including amylin, corticotropin releasing factor (CRF) and ghrelin were not differentially expressed between lines, while mRNA abundance of calcitonin, CRF receptor 1, leptin receptor, neuropeptide S, melanocortin receptor 3, and oxytocin were greater in LWS than HWS chicks. Pro-opiomelanocortin mRNA was lower in LWS than HWS chicks, while fasting decreased its expression in both lines. These results suggest that there are differences in gene expression of appetite-associated factors between LWS and HWS lines that might be associated with their differential food intake and thus contribute to differences in severity of anorexia, body weight, adiposity, and development of obesity. PMID:25677648

  2. Peptides and Food Intake

    PubMed Central

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  3. Peptides and food intake.

    PubMed

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  4. Expression of Caenorhabditis elegans antimicrobial peptide NLP-31 in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Lim, Mei-Perng; Nathan, Sheila

    2014-09-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a fulminant disease endemic in Southeast Asia and Northern Australia. The standardized form of therapy is antibiotics treatment; however, the bacterium has become increasingly resistant to these antibiotics. This has spurred the need to search for alternative therapeutic agents. Antimicrobial peptides (AMPs) are small proteins that possess broad-spectrum antimicrobial activity. In a previous study, the nematode Caenorhabditis elegans was infected by B. pseudomallei and a whole animal transcriptome analysis identified a number of AMP-encoded genes which were induced significantly in the infected worms. One of the AMPs identified is NLP-31 and to date, there are no reports of anti-B. pseudomallei activity demonstrated by NLP-31. To produce NLP-31 protein for future studies, the gene encoding for NLP-31 was cloned into the pET32b expression vector and transformed into Escherichia coli BL21(DE3). Protein expression was induced with 1 mM IPTG for 20 hours at 20°C and recombinant NLP-31 was detected in the soluble fraction. Taken together, a simple optimized heterologous production of AMPs in an E. coli expression system has been successfully developed.

  5. Recombinant expression and biological characterization of the antimicrobial peptide fowlicidin-2 in Pichia pastoris

    PubMed Central

    Xing, Li-Wei; Tian, Shi-Xun; Gao, Wei; Yang, Na; Qu, Pei; Liu, Di; Jiao, Jian; Wang, Jue; Feng, Xing-Jun

    2016-01-01

    Fowlicidins are a group of cathelicidin antimicrobial peptides that were initially identified in chickens. Fowlicidin-2, which is composed of 31 amino acids, is widely expressed in the majority of tissues in chickens and has an important role in innate immunity. In the present study, a recombinant expression system for fowlicidin-2 was successfully constructed using Pichia pastoris X-33 and the expression vector pPICZα-A. Under the optimized fermentation conditions, 85.6 mg fowlicidin-2 with >95% purity was obtained from 1 liter culture medium following purification by ion exchange chromatography and reversed phase high performance liquid chromatography. The recombinant fowlicidin-2 exhibited broad spectrum antimicrobial activity and had a minimum inhibitory concentration ranging from 1 to 4 µM. Furthermore, recombinant fowlicidin-2 exhibited hemolytic activity, promoting 50% human erythrocyte hemolysis in the concentration range of 128–256 µM, and anticancer activity, resulting in the death of 50% of A375 human malignant melanoma cells in the concentration range of 2–4 µM. The results of the present study suggest that recombinant fowlicidin-2 may be a promising candidate for therapeutic applications. PMID:27698732

  6. Recombinant expression and biological characterization of the antimicrobial peptide fowlicidin-2 in Pichia pastoris

    PubMed Central

    Xing, Li-Wei; Tian, Shi-Xun; Gao, Wei; Yang, Na; Qu, Pei; Liu, Di; Jiao, Jian; Wang, Jue; Feng, Xing-Jun

    2016-01-01

    Fowlicidins are a group of cathelicidin antimicrobial peptides that were initially identified in chickens. Fowlicidin-2, which is composed of 31 amino acids, is widely expressed in the majority of tissues in chickens and has an important role in innate immunity. In the present study, a recombinant expression system for fowlicidin-2 was successfully constructed using Pichia pastoris X-33 and the expression vector pPICZα-A. Under the optimized fermentation conditions, 85.6 mg fowlicidin-2 with >95% purity was obtained from 1 liter culture medium following purification by ion exchange chromatography and reversed phase high performance liquid chromatography. The recombinant fowlicidin-2 exhibited broad spectrum antimicrobial activity and had a minimum inhibitory concentration ranging from 1 to 4 µM. Furthermore, recombinant fowlicidin-2 exhibited hemolytic activity, promoting 50% human erythrocyte hemolysis in the concentration range of 128–256 µM, and anticancer activity, resulting in the death of 50% of A375 human malignant melanoma cells in the concentration range of 2–4 µM. The results of the present study suggest that recombinant fowlicidin-2 may be a promising candidate for therapeutic applications.

  7. Regulation of growth-blocking peptide expression during embryogenesis of the cabbage army worm

    SciTech Connect

    Tsuzuki, Seiji; Sekiguchi, Shiroh; Hayakawa, Yoichi . E-mail: hayakayo@cc.saga-u.ac.jp

    2005-10-07

    Growth-blocking peptide (GBP) is an insect cytokine with diverse biological functions. Northern blot analysis revealed high heterogeneity in the size distribution of GBP mRNAs as well as in the tissues where they are detected. The s patio-temporal transcription pattern is dynamic, especially during embryogenesis. Gel shift assays demonstrated that the cabbage army worm embryo nuclear extract specifically binds to a 178-bp element, at position +234 to +411 from the transcription start site of the 1.3 kb GBP transcript, in which two Drosophila Deformed (DfD) binding sites are repeated in tandem. The specific binding between this element and Dfd was demonstrated using recombinant cabbage armyworm Dfd protein. Silencing the Dfd expression in embryos by treating with DfD double-stranded RNA did not reduce the expression level of GBP, but ectopic GBP expression was observed in the lateral region of the embryo, suggesting that DD could serve as a transcriptional repressor for the GBP gene.

  8. Diurnal profiles of hypothalamic energy balance gene expression with photoperiod manipulation in the Siberian hamster, Phodopus sungorus.

    PubMed

    Ellis, Claire; Moar, Kim M; Logie, Tracy J; Ross, Alexander W; Morgan, Peter J; Mercer, Julian G

    2008-04-01

    Hypothalamic energy balance genes have been examined in the context of seasonal body weight regulation in the Siberian hamster. Most of these long photoperiod (LD)/short photoperiod (SD) comparisons have been of tissues collected at a single point in the light-dark cycle. We examined the diurnal expression profile of hypothalamic genes in hamsters killed at 3-h intervals throughout the light-dark cycle after housing in LD or SD for 12 wk. Gene expression of neuropeptide Y, agouti-related peptide, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, long-form leptin receptor, suppressor of cytokine signaling-3, melanocortin-3 receptor, melanocortin-4 receptor, and the clock gene Per1 as control were measured by in situ hybridization in hypothalamic nuclei. Effects of photoperiod on gene expression and leptin levels were generally consistent with previous reports. A clear diurnal variation was observed for Per1 in the suprachiasmatic nucleus in both photoperiods. Temporal effects on expression of energy balance genes were restricted to long-form leptin receptor in the arcuate nucleus and ventromedial nucleus, where similar diurnal expression profiles were observed, and melanocortin-4 receptor in the paraventricular nucleus; these effects were only observed in LD hamsters. There was no variation in serum leptin concentration. The 24-h profiles of hypothalamic energy balance gene expression broadly confirm photoperiodic differences that were observed previously, based on single time point comparisons, support the growing consensus that these genes have a limited role in seasonal body weight regulation, and further suggest limited involvement in daily rhythms of food intake.

  9. Seasonal Differences in Relative Gene Expression of Putative Central Appetite Regulators in Arctic Charr (Salvelinus alpinus) Do Not Reflect Its Annual Feeding Cycle

    PubMed Central

    Striberny, Anja; Ravuri, Chandra Sekhar; Jobling, Malcolm; Jørgensen, Even Hjalmar

    2015-01-01

    The highly seasonal anadromous Arctic charr (Salvelinus alpinus) was used to investigate the possible involvement of altered gene expression of brain neuropeptides in seasonal appetite regulation. Pro-opiomelanocortin (POMCA1, POMCA2), Cocaine and amphetamine regulated transcript (CART), Agouti related Peptide (AgRP), Neuropeptide Y (NPY) and Melanocortin Receptor 4 (MC4-R) genes were examined. The function of centrally expressed Leptin (Lep) in fish remains unclear, so Lep (LepA1, LepA2) and Leptin Receptor (LepR) genes were included in the investigation. In a ten months study gene expression was analysed in hypothalamus, mesencephalon and telencephalon of immature charr held under natural photoperiod (69°38’N) and ambient temperature and given excess feed. From April to the beginning of June the charr did not feed and lost weight, during July and August they were feeding and had a marked increase in weight and condition factor, and from November until the end of the study the charr lost appetite and decreased in weight and condition factor. Brain compartments were sampled from non-feeding charr (May), feeding charr (July), and non-feeding charr (January). Reverse transcription real-time quantitative PCR revealed temporal patterns of gene expression that differed across brain compartments. The non-feeding charr (May, January) had a lower expression of the anorexigenic LepA1, MC4-R and LepR in hypothalamus and a higher expression of the orexigenic NPY and AgRP in mesencephalon, than the feeding charr (July). In the telencephalon, LepR was more highly expressed in January and May than in July. These results do not indicate that changes in central gene expression of the neuropeptides investigated here directly induce seasonal changes in feeding in Arctic charr. PMID:26421838

  10. Leucocyte expression of genes implicated in the plasminogen activation cascade is modulated by yoghurt peptides.

    PubMed

    Theodorou, Georgios; Politis, Ioannis

    2016-08-01

    The urokinase-plasminogen activator (u-PA), its receptor (u-PAR) and the inhibitors of u-PA (PAI-1 and PAI-2) provide a multi-molecular system in leucocytes that exerts pleiotropic functions influencing the development of inflammatory and immune responses. The objective of the present study was to examine the ability of water soluble extracts (WSE) obtained from traditional Greek yoghurt made from bovine or ovine milk to modulate the expression of u-PA, u-PAR, PAI-1 and PAI-2 in ovine monocytes and neutrophils. WSE were obtained from 8 commercial traditional type Greek yoghurts made from ovine or bovine milk. WSE upregulated the expression of all 4 u-PA related genes in monocytes but the upregulation was much higher in the PAI-1 (10-fold) than in u-PA and u-PAR (3-4 fold) thus, shifting the system towards inhibition. In line with this observation, WSE reduced total and membrane-bound u-PA activity in monocytes. In neutrophils, WSE caused small (50-60%) but significant (P < 0·05) reductions in expression of u-PAR and PAI-2 but had no effect on expression of u-PA, PAI-1 and on total cell-associated and membrane-bound u-PA activity. WSE from yoghurts made from bovine or ovine milk were essentially equally effective in affecting the u-PA system except for the u-PAR gene in ovine neutrophils that was affected (reduced) by the ovine and not the bovine WSE. In conclusion, peptides present in WSE modulated the expression of u-PA related genes but the effect was much more prominent in monocytes than in neutrophils. PMID:27600972

  11. Regulation of tracheal antimicrobial peptide gene expression in airway epithelial cells of cattle.

    PubMed

    Taha-Abdelaziz, Khaled; Wyer, Leanna; Berghuis, Lesley; Bassel, Laura L; Clark, Mary Ellen; Caswell, Jeff L

    2016-01-01

    β-defensins are an important element of the mucosal innate immune response against bacterial pathogens. Tracheal antimicrobial peptide (TAP) has microbicidal activity against the bacteria that cause bovine respiratory disease, and its expression in tracheal epithelial cells is upregulated by bacterial products including lipopolysaccharide (LPS, a TLR4 agonist), Pam3CSK4 (an agonist of Toll-like receptor 2/1), and interleukin (IL)-17A. The objectives of this study were to identify the signalling pathway by which LPS, Pam3CSK4 and IL-17A induce TAP gene expression, and to determine the effect of glucocorticoid as a model of stress on this epithelial innate immune response. In primary cultures of bovine tracheal epithelial cells (bTEC), LPS, Pam3CSK4 and IL-17A each stimulated TAP gene expression. This effect was abrogated by caffeic acid phenylester (CAPE), an inhibitor of NF-κB. Similarly, western analysis showed that LPS, Pam3CSK4 and IL-17A each induced translocation of NF-κB p65 from the cytoplasm to the nucleus, but pre-treatment with CAPE inhibited this response. Finally, pre-treatment of bTEC with the glucocorticoid dexamethasone abolished the stimulatory effect of LPS, Pam3CSK4 and IL-17A on upregulation of TAP gene expression. These findings indicate that NF-κB activation is necessary for induction of TAP gene expression by LPS (a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist), or IL-17A. Furthermore, this stimulatory response is inhibited by glucocorticoid, suggesting this as one mechanism by which stress increases the risk of bacterial pneumonia. These findings have implications for understanding the pathogenesis of stress-associated bacterial pneumonia, and for developing methods to stimulate innate immune responses in the respiratory tract of cattle. PMID:26987959

  12. Expression of an antimicrobial peptide, digestive enzymes and nutrient transporters in the intestine of E. praecox-infected chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters and an antimicrobial peptide following an Eimeria praecox challenge of chickens at d...

  13. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system.

    PubMed

    Pechsrichuang, Phornsiri; Songsiriritthigul, Chomphunuch; Haltrich, Dietmar; Roytrakul, Sittiruk; Namvijtr, Peenida; Bonaparte, Napolean; Yamabhai, Montarop

    2016-01-01

    The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli. PMID:27516938

  14. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system.

    PubMed

    Pechsrichuang, Phornsiri; Songsiriritthigul, Chomphunuch; Haltrich, Dietmar; Roytrakul, Sittiruk; Namvijtr, Peenida; Bonaparte, Napolean; Yamabhai, Montarop

    2016-01-01

    The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli.

  15. H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

    PubMed

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko; Akagawa, Mitsugu; Tsuji-Naito, Kentaro

    2016-07-01

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. PMID:27216463

  16. Modulation of chicken intestinal immune gene expression by small cationic peptides as feed additives during the first week posthatch.

    PubMed

    Kogut, Michael H; Genovese, Kenneth J; He, Haiqi; Swaggerty, Christina L; Jiang, Yiwei

    2013-09-01

    We have been investigating modulation strategies tailored around the selective stimulation of the host's immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soil bacterium, Brevibacillus texasporus. These peptides have immune modulatory properties that enhance both leukocyte functional efficiency and leukocyte proinflammatory cytokine and chemokine mRNA transcription activities in vitro. In addition, when provided as a feed additive for just 4 days posthatch, BT peptides significantly induce a concentration-dependent protection against cecal and extraintestinal colonization by Salmonella enterica serovar Enteritidis. In the present studies, we assessed the effects of feeding BT peptides on transcriptional changes on proinflammatory cytokines, inflammatory chemokines, and Toll-like receptors (TLR) in the ceca of broiler chickens with and without S. Enteritidis infection. After feeding a BT peptide-supplemented diet for the first 4 days posthatch, chickens were then challenged with S. Enteritidis, and intestinal gene expression was measured at 1 or 7 days postinfection (p.i.) (5 or 11 days of age). Intestinal expression of innate immune mRNA transcripts was analyzed by quantitative real-time PCR (qRT-PCR). Analysis of relative mRNA expression showed that a BT peptide-supplemented diet did not directly induce the transcription of proinflammatory cytokine, inflammatory chemokine, type I/II interferon (IFN), or TLR mRNA in chicken cecum. However, feeding the BT peptide-supplemented diet primed cecal tissue for increased (P ≤ 0.05) transcription of TLR4, TLR15, and TLR21 upon infection with S. Enteritidis on days 1 and 7 p.i. Likewise, feeding the BT peptides primed the cecal tissue for increased transcription of proinflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-18, type I and II IFNs) and inflammatory chemokine (CxCLi2

  17. Sex, offspring and carcass determine antimicrobial peptide expression in the burying beetle

    PubMed Central

    Jacobs, Chris G. C.; Steiger, Sandra; Heckel, David G.; Wielsch, Natalie; Vilcinskas, Andreas; Vogel, Heiko

    2016-01-01

    The burying beetle Nicrophorus vespilloides has emerged as a model system for the investigation of adaptations that allow the utilization of carrion as a diet and as a resource for reproduction. The survival of beetles and their offspring given their exposure to soil-dwelling and cadaver-borne microbes requires mechanisms that reduce bacterial contamination in the diet and that achieve sanitation of the microhabitat. To explore the role of antimicrobial peptides (AMPs) in this context, we analyzed burying beetle males and females at different stages of their breeding cycle using the RNA-Seq and proteomics approaches. To address variation in immune functions, we investigated the impact of adult sex, the presence or absence of offspring (social context), and the presence of carrion (environmental context) on the expression of the identified immune effector genes. We found that particular AMPs are sex-specific and tightly regulated by the presence of a carcass or offspring and identified the two most context-dependent antimicrobial proteins in anal secretions. The context-specific expression dynamics of particular AMPs and lysozymes reveals a complex regulatory system, reflecting adaptations to specific ecological niches. This study highlights how burying beetles cope with microorganisms found on carrion and identifies candidates for both internal and external immunity. PMID:27139635

  18. Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

    PubMed

    Pelc, Rebecca S; McClure, Jennifer C; Kaur, Simran J; Sears, Khandra T; Rahman, M Sayeedur; Ceraul, Shane M

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria's ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.

  19. Induced expression of neuronal membrane attack complex and cell death by Alzheimer's beta-amyloid peptide.

    PubMed

    Shen, Y; Sullivan, T; Lee, C M; Meri, S; Shiosaki, K; Lin, C W

    1998-06-15

    beta-amyloid peptide (A beta) and complement-derived membrane attack complex (MAC) are co-localized in senile plaques of brains from Alzheimer's disease (AD) patients. But the relationship between A beta and complement activation is unclear. We have used human neurotypic cells, differentiated SH-SY5Y, as a model system to examine regulation of neuronal MAC expression and cell death by A beta. We demonstrated that mRNAs (C1q, C2, C3, C4, C5, C6, C7, C8 and C9) and proteins (C1q, C3 and C9) for the major components of the classical complement cascade are present in the SH-SY5Y neurotypic cells, indicating that neuronal cells can synthesize the necessary proteins required for MAC formation. Furthermore, immunocytochemical studies showed the A beta-induced neuronal MAC expression on the SH-SY5Y cells after CD59 was removed by PIPLC or blocked by anti-CD59 antibody. Meanwhile, increased A beta-induced neuronal cell death was observed following treatment with anti-CD59. Taken together, these results suggest that A beta activates neuronal complement cascade to induce MAC, and a deficiency of endogenous complement regulatory proteins, e.g., CD59, may increase the vulnerability of neurons to complement-mediated cytotoxicity. PMID:9689469

  20. [Expression, purification of recombinant cationic peptide AIK in Escherichia coli and its antitumor activity].

    PubMed

    Fan, Fangfang; Sun, Huiying; Xu, Hui; Liu, Jiawei; Zhang, Haiyuan; Li, Yilan; Ning, Xuelian; Sun, Yue; Bai, Jing; Fu, Songbin; Zhou, Chunshui

    2015-12-01

    AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future. PMID:27093838

  1. Expression of antimicrobial peptide genes in Bombyx mori gut modulated by oral bacterial infection and development.

    PubMed

    Wu, Shan; Zhang, Xiaofeng; He, Yongqiang; Shuai, Jiangbing; Chen, Xiaomei; Ling, Erjun

    2010-11-01

    Although Bombyx mori systematic immunity is extensively studied, little is known about the silkworm's intestine-specific responses to bacterial infection. Antimicrobial peptides (AMPs) gene expression analysis of B. mori intestinal tissue to oral infection with the Gram-positive (Staphylococcus aureus) and -negative (Escherichia coli) bacteria revealed that there is specificity in the interaction between host immune responses and parasite types. Neither Att1 nor Leb could be stimulated by S. aureus and E. coli. However, CecA1, Glo1, Glo2, Glo3, Glo4 and Lys, could only be trigged by S. aureus. On the contrary, E. coli stimulation caused the decrease in the expression of CecA1, Glo3 and Glo4 in some time points. Interestingly, there is regional specificity in the silkworm local gut immunity. During the immune response, the increase in Def, Hem and LLP3 was only detected in the foregut and midgut. For CecB1, CecD, LLP2 and Mor, after orally administered with E. coli, the up-regulation was only limited in the midgut and hindgut. CecE was the only AMP that positively responses to the both bacteria in all the testing situations. With development, the expression levels of the AMPs were also changed dramatically. That is, at spinning and prepupa stages, a large increase in the expression of CecA1, CecB1, CecD, CecE, Glo1, Glo2, Glo3, Glo4, Leb, Def, Hem, Mor and Lys was detected in the gut. Unexpectedly, in addition to the IMD pathway genes, the Toll and JAK/STAT pathway genes in the silkworm gut can also be activated by microbial oral infection. But in the developmental course, corresponding to the increase in expression of AMPs at spinning and prepupa stages, only the Toll pathway genes in the gut exhibit the similar increasing trend. Our results imply that the immune responses in the silkworm gut are synergistically regulated by the Toll, JAK/STAT and IMD pathways. However, as the time for approaching pupation, the Toll pathway may play a role in the AMPs expression

  2. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  3. Adipose tissue natriuretic peptide receptor expression is related to insulin sensitivity in obesity and diabetes

    PubMed Central

    Kovacova, Zuzana; Tharp, William G.; Liu, Dianxin; Wei, Wan; Xie, Hui

    2016-01-01

    Objective Cardiac natriuretic peptides (NPs) bind to two receptors (NPRA‐mediator of signaling; NPRC‐clearance receptor) whose ratio, NPRR (NPRA/NPRC), determines the NP bioactivity. This study investigated the relationship of NP receptor gene expression in adipose tissue and muscle with obesity and glucose intolerance. Prospectively, the study also assessed whether changes in NP receptor expression and thermogenic gene markers accompanied improvements of insulin sensitivity. Methods A cross‐sectional study of subjects with a wide range of BMI and glucose tolerance (n = 50) was conducted, as well as a randomized 12‐week trial of subjects with type 2 diabetes mellitus (T2DM) treated with pioglitazone (n = 9) or placebo (n = 10). Results NPRR mRNA was significantly lower in adipose tissue of subjects with obesity when compared with lean subjects (P ≤ 0.001). NPRR decreased with progression from normal glucose tolerance to T2DM (P < 0.01) independently of obesity. Treatment of subjects with T2DM with pioglitazone increased NPRR in adipose tissue (P ≤ 0.01) in conjunction with improvements in insulin sensitivity and increases of the thermogenic markers PPARγ coactivator‐1α and uncoupling protein 1 (P ≤ 0.01). Conclusions Decreased adipose tissue NPRR was associated with obesity, glucose intolerance, and insulin resistance. This relationship was not observed for skeletal muscle NPRR. Pharmacological improvement of insulin sensitivity in subjects with T2DM was tied to improvement in NPRR and increased expression of genes involved in thermogenic processes. PMID:26887289

  4. [Preparation of polyacrylamide gel electrophoresis for human chorionic gonadotropin chimeric peptide 12 expressed in E. coli].

    PubMed

    Zou, Yong-Shui; Xu, Wan-Xiang; He, Yuan; Sun, Zhi-Da; Xue, Xiao-Lin

    2002-09-01

    In recent years, development of chimeric peptide (CP) immunogens is a trend in the vaccinological field. The CPs contain a B cell epitope(s) of target antigen and a promiscuous self - or foreign- T cell epitope(s). However, such constructed CPs were all expressed in prokaryotic or eukaryotic systems at lower levels. To purify the human chorionic gonadotropin (hCG) CP12 expressed in E. coli at the level of about 1% of the total cell proteins, an improved method of preparative gel polyacrylamide gel electrophoresis (PAGE) was developed. The important improvement to routine preparative PAGE involves: (1) running reversed electrophoresis by rearranging the gel- carrying plate when the bromophenol blue band arrived at 1-1.5 centimeter from the bottom of the gel; (2) making a collecting trough between the gel and a dialytic membrane that was used to isolate the upper tank buffer. About 8 fractions were collected at regular intervals of 15 minutes after bromophenol blue running out of gel. And then 0.2 ml was taken from each fraction and the protein was precipitated by sequentially adding trichloroacetic acid and acetone. Each sample was dissolved in 20 microL sample buffer and analyzed and identified by SDS-PAGE and Western blotting. As a result, the hCG CP12 expression product with 95% relative homogeneity was harvested at a 50-100 microgram level after a single-step purification of this preparative PAGE, with respect to the sample which contained 3-4 mg of cell proteins. PMID:12198575

  5. Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization.

    PubMed

    Wei, Xuetuan; Zhou, Yinhua; Chen, Jingbang; Cai, Dongbo; Wang, Dan; Qi, Gaofu; Chen, Shouwen

    2015-02-01

    Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

  6. Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord

    PubMed Central

    Barry, Devin M; Li, Hui; Liu, Xian-Yu; Shen, Kai-Feng; Liu, Xue-Ting; Wu, Zhen-Yu; Munanairi, Admire; Chen, Xiao-Jun; Yin, Jun; Sun, Yan-Gang; Li, Yun-Qing

    2016-01-01

    There are substantial disagreements about the expression of gastrin-releasing peptide (GRP) in sensory neurons and whether GRP antibody cross-reacts with substance P (SP). These concerns necessitate a critical revaluation of GRP expression using additional approaches. Here, we show that a widely used GRP antibody specifically recognizes GRP but not SP. In the spinal cord of mice lacking SP (Tac1 KO), the expression of not only GRP but also other peptides, notably neuropeptide Y (NPY), is significantly diminished. We detected Grp mRNA in dorsal root ganglias using reverse transcription polymerase chain reaction, in situ hybridization and RNA-seq. We demonstrated that Grp mRNA and protein are upregulated in dorsal root ganglias, but not in the spinal cord, of mice with chronic itch. Few GRP+ immunostaining signals were detected in spinal sections following dorsal rhizotomy and GRP+ cell bodies were not detected in dissociated dorsal horn neurons. Ultrastructural analysis further shows that substantially more GRPergic fibers form synaptic contacts with gastrin releasing peptide receptor-positive (GRPR+) neurons than SPergic fibers. Our comprehensive study demonstrates that a majority of GRPergic fibers are of primary afferent origin. A number of factors such as low copy number of Grp transcripts, small percentage of cells expressing Grp, and the use of an eGFP GENSAT transgenic as a surrogate for GRP protein have contributed to the controversy. Optimization of experimental procedures facilitates the specific detection of GRP expression in dorsal root ganglia neurons. PMID:27068287

  7. Expression of messenger RNAs for peptides and tyrosine hydroxylase in primary sensory neurons that innervate arterial baroreceptors and chemoreceptors.

    PubMed

    Czyzyk-Krzeska, M F; Bayliss, D A; Lawson, E E; Millhorn, D E

    1991-08-01

    Retrograde fiber tracing and in situ hybridization were used to determine expression of mRNAs for preprotachykinin A (ppTA), calcitonin gene related peptide (CGRP), preproenkephalin A (ENK), neuropeptide tyrosine (NPY) and somatostatin (SOM) as well as tyrosine hydroxylase (TH) in the petrosal ganglia primary sensory neurons which innervate carotid sinus baroreceptors and carotid body chemoreceptors. Perfusion of the carotid sinus with the retrogradely transported dye (Fluoro-Gold) labeled primary sensory neurons in petrosal ganglion. Numerous somata in the petrosal ganglion labeled with dye contained mRNAs for all the above peptides, except SOM. Moreover, TH mRNA was found in a substantial number of retrogradely labeled cells in the petrosal ganglion. This study provides information concerning which of the numerous peptides identified in sensory neurons of petrosal ganglion may be involved in modulation of the arterial baroreceptor and chemoreceptor reflexes. PMID:1681484

  8. Differential effect of prolonged food restriction and fasting on hypothalamic malonyl-CoA concentration and expression of orexigenic and anorexigenic neuropeptides genes in rats.

    PubMed

    Sucajtys-Szulc, Elzbieta; Turyn, Jacek; Goyke, Elzbieta; Korczynska, Justyna; Stelmanska, Ewa; Slominska, Ewa; Smolenski, Ryszard T; Rutkowski, Boleslaw; Swierczynski, Julian

    2010-02-01

    Several lines of evidence suggest that malonyl-CoA in the hypothalamus plays an important role in monitoring and modulating body energy balance. In fasted state the level of malonyl-CoA concentration significantly decreases. Simultaneously, orexigenic neuropeptides (NPY - neuropeptide Y, AgRP - agouti-related peptide) genes are expressed at high level, whereas anorexigenic neuropeptides (CART - cocaine-and amphetamine-regulated transcript, POMC - proopiomelanocortin) genes are expressed at low level. When food intake resumes, opposite effect is observed. This study examined the effect of prolonged food restriction, common in humans trying to lose body weight on expression of orexigenic and anorexigenic neuropeptides genes and on malonyl-CoA content in rat whole hypothalamus. We observed an increase of NPY and AgRP mRNA levels in hypothalamus of rats kept on 30 days-long food restriction (50% of the amount of food consumed by controls). Simultaneously, a decrease of CART and POMC mRNA levels occurred. Refeeding caused a decrease in NPY and POMC mRNA levels without effect on AgRP and CART mRNA. Surprisingly, both prolonged food restriction and food restriction/refeeding caused the increase of malonyl-CoA level in whole hypothalamus. In contrast, fasting for 24h caused the decrease of malonyl-CoA level, which was associated with the up-regulation of NPY and AgRP genes expression and down-regulation of CART and POMC genes expression. After refeeding opposite effect was observed. These results indicate that prolonged food restriction and acute fasting, conditions in which energy expenditure exceeds intake, differentially affect malonyl-CoA concentration and similarly affect orexigenic and anorexigenic neuropeptide genes expression in whole rat hypothalamus.

  9. Variations in the expressed antimicrobial peptide repertoire of northern leopard frog (Rana pipiens) populations suggest intraspecies differences in resistance to pathogens

    PubMed Central

    Tennessen, Jacob A.; Woodhams, Douglas C.; Chaurand, Pierre; Reinert, Laura K.; Billheimer, Dean; Shyr, Yu; Caprioli, Richard M.; Blouin, Michael S.; Rollins-Smith, Louise A.

    2010-01-01

    The northern leopard frog (Rana pipiens or Lithobates pipiens) is historically found in most of the provinces of Canada and the northern and southwest states of the United States. In the last 50 years, populations have suffered significant losses, especially in the western regions of the species range. Using a peptidomics approach, we show that the pattern of expressed antimicrobial skin peptides of frogs from three geographically separated populations are distinct, and we report the presence of four peptides (brevinin-1Pg, brevinin-1Pl, ranatuerin-2Pb, and ranatuerin-2Pc) that have not previously been found in skin secretions. The differences in expressed peptides reflect differences in the distribution of alleles for the newly described Brevinin1.1 locus in the three populations. When enriched peptide mixtures were tested for their ability to inhibit growth of the pathogenic amphibian chytrid (Batrachochytrium dendrobatidis), peptides from Minnesota or Vermont frogs were more effective that peptides from Michigan frogs. Four of the purified peptides were tested for their ability to inhibit growth of two bacterial pathogens (Aeromonas hydrophila and Staphylococcus epidermidis) and B. dendrobatidis. Three of the four were effective inhibitors of B. dendrobatidis and S. epidermidis, but none inhibited A. hydrophila. We interpret these differences in expression and activity of antimicrobial peptides as evidence to suggest that each population may have been selected to express a suite of peptides that reflects current and past encounters with skin microbes. PMID:19622371

  10. A synthetic peptide derived from the D1 domain of flagellin induced the expression of proinflammatory cytokines in fish macrophages.

    PubMed

    González-Stegmaier, Roxana; Guzmán, Fanny; Albericio, Fernando; Villarroel-Espíndola, Franz; Romero, Alex; Mulero, Victoriano; Mercado, Luis

    2015-11-01

    Flagellin is the main protein component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in fish and mammals. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines, and other immunological functions. We have previously reported that recombinant flagellin from Vibrio anguillarum and its ND1 domain are able to upregulate the expression of genes encoding major the proinflammatory mediators in gilthead seabream and rainbow trout macrophages. Considering the key role of D1 domain of flagellin for binding to TLR5M and its immunostimulatory activity, we designed and chemically synthesized a peptide derived of this region. The effects of the synthetic peptide were evaluated in vitro using head kidney macrophages from gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species the expression of genes encoding the proinflammatory cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), and the chemokine IL-8, was induced upon stimulation of macrophages with the D1 domain synthetic peptide. IL-1β and IL-8 were the most upregulated genes and to a lesser extent TNF-α. Interestingly, however, the induction activity of the synthetic peptide was higher in gilthead seabream than in rainbow trout macrophages. The results were confirmed at the protein levels for IL-8. Collectively, these results suggest that synthetic peptide derived from flagelling could be a promising approach for the immunostimulation and vaccination of farmed fish. PMID:26363237

  11. Recombinant expression and downstream processing of the disulfide-rich tumor-targeting peptide chlorotoxin.

    PubMed

    Wang, Xiao-Min; Luo, Xiao; Guo, Zhan-Yun

    2013-10-01

    Chlorotoxin (CTX) is a scorpion-derived disulfide-rich peptide that targets malignant tumors by binding the cell surface matrix metalloproteinase-2 and annexin A2. Various CTXs labeled with functional moieties have shown great potential for tumor diagnosis and treatment. In the present study, we established an efficient approach for preparing mature CTX that may be used for experimental and therapeutic purposes. The designed CTX precursors carried either a 6xHis-tag or a 6xHis-tag and a glutathione transferase (GST)-tag and were recombinantly expressed in Escherichia coli. Following S-sulfonation, the precursors were purified using immobilized metal-ion affinity chromatography. Subsequent to the removal of the tag by enterokinase cleavage and in vitro oxidative refolding, mature CTX was obtained with a considerable yield. The yield of mature CTX whose precursors carried a 6xHis-tag and a GST-tag (2 mg per liter of culture) was ∼10-fold that of the mature CTX whose precursors carried a 6xHis-tag (150-200 μg per liter of culture). The folded CTX inhibited the migration of glioma cells in a concentration-dependent manner, suggesting it was biologically active.

  12. Generation and analysis of lentivirus expressing a 2A peptide-linked bicistronic fluorescent construct.

    PubMed

    Huss, David; Lansford, Rusty

    2014-12-01

    This weeklong protocol for making and testing lentivirus has been used in the Advanced Topics in Molecular Neuroscience (ATMN) lecture and laboratory course at Cold Spring Harbor Laboratory (CSHL) for nearly a decade. Lentiviruses are derived from HIV-1 and are ideal vehicles for the delivery of multiple genes of interest into target cells. In this protocol, 2A peptide-linked sequences are used to create a bicistronic lentiviral construct containing a ubiquitous promoter (chick β actin with a cytomegalovirus [CMV] early enhancer) driving dual expression of two fluorescent proteins (FP): H2B-Cerulean (a nuclear-localized blue FP) and Dendra2 (a photoactivatable green FP that converts to red after exposure to UV light). Polymerase chain reaction (PCR) amplification of the bicistronic insert is followed by subcloning into a lentiviral vector and transfection into a packaging cell line. The resulting viral supernatants can be used to prepare concentrated stocks and infect cells for imaging via epifluorescent and confocal microscopy. PMID:25447284

  13. Culture on Electrospun Polyurethane Scaffolds Decreases Atrial Natriuretic Peptide Expression by Cardiomyocytes In Vitro

    PubMed Central

    Rockwood, Danielle N.; Akins, Robert E.; Parrag, Ian; Woodhouse, Kimberly A.; Rabolt, John F.

    2008-01-01

    The function of the mammalian heart depends on the functional alignment of cardiomyocytes, and controlling cell alignment is an important consideration in biomaterial design for cardiac tissue engineering and research. The physical cues that guide functional cell alignment in vitro and the impact of substrate-imposed alignment on cell phenotype, however, are only partially understood. In this report, primary cardiac ventricular cells were grown on electrospun, biodegradable polyurethane (ES-PU) with either aligned or unaligned microfibers. ES-PU scaffolds supported high-density cultures, and cell subpopulations remained intact over two weeks in culture. ES-PU cultures contained electrically-coupled cardiomyocytes with connexin-43 localized to points of cell:cell contact. Multi-cellular organization correlated with microfiber orientation, and aligned materials yielded highly oriented cardiomyocyte groupings. Atrial natriuretic peptide, a molecular marker that has decreasing expression during ventricular cell maturation, was significantly lower in cultures grown on ES-PU scaffolds than in those grown on tissue culture polystyrene. Cells grown on aligned ES-PU had significantly lower steady state levels of ANP and constitutively released less ANP over time indicating that scaffold-imposed cell organization resulted in a shift in cell phenotype to a more mature state. We conclude that the physical organization of microfibers in ES-PU scaffolds impacts both multi-cellular architecture and cardiac cell phenotype in vitro. PMID:18823659

  14. Higher efficiency soluble prokaryotic expression, purification, and structural analysis of antimicrobial peptide G13.

    PubMed

    Che, Yuanyuan; Lu, Yinghu; Zha, Xiangdong; Huang, Huoqing; Yang, Peilong; Ma, Lijuan; Xu, Xuejiao

    2016-03-01

    G13 is a 19-residue cationic antimicrobial peptide derived from granulysin. In order to achieve high-level expression of G13 in Escherichia coli cells, and to reduce downstream processing costs, we introduced an Asp-Pro acid labile bond between the His-Patch thioredoxin and G13 and constructed the recombinant plasmid pThiohisA-DP-G13. The plasmid was transformed into E. coli BL21 (DE3). After induction with isopropyl-β-d-thiogalactopyranoside for 5 h, the fusion protein accumulated up to 200 mg/L in soluble form. The fusion protein was released by a high pressure homogenizer, cleaved using 13% acetic acid at 50 °C hydrolysis for 72 h. The recombinant G13 (r-G13) was then successively purified by fractional precipitation with ammonium sulfate and trichloroacetic acid, followed by one-step cation exchange chromatography. The purified r-G13 displayed a single band (about 2.2 kDa) as analyzed by Tris-Tricine buffered SDS-PAGE, and its precise molecular weight was confirmed using tandem mass spectrometry. Analysis of r-G13 by circular dichroism (CD) indicated that r-G13 contained predominantly β-sheet and random coil. Agar plate diffusion assay revealed that the r-G13 exhibited antibacterial activity against both Bacillus subtilis and E. coli.

  15. Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium

    PubMed Central

    Geldart, Kathryn; Borrero, Juan

    2015-01-01

    Antibiotic-resistant enterococcal infections are a major concern in hospitals where patients with compromised immunity are readily infected. Enterococcus faecium bacteria are of particular interest as these pathogens account for over 80% of vancomycin-resistant enterococcal infections. Antimicrobial peptides (AMPs) produced at the site of infection by engineered bacteria may offer a potential alternative to traditional antibiotics for the treatment of resistant bacteria such as E. faecium. For this mode of delivery to be effective, it is essential to identify a suitable protein expression system that can be used in the desired delivery bacterium. In this study, we describe a promising chloride-inducible promoter and its application in the bacterial delivery of AMPs from Lactococcus lactis to reduce counts of E. faecium bacteria in vitro. Reporter gene studies show that at chloride concentrations found within the human intestines, the chloride-inducible promoter exhibits high levels of protein expression compared to those of the commonly used nisin-inducible promoter. These results indicate that this system is powerful and would not require the exogenous administration of an inducer molecule. In its application for AMP production against E. faecium in vitro, L. lactis producing AMPs under the chloride promoter rapidly decreased E. faecium counts by nearly 10,000-fold. As an extension of this application, we also demonstrate the potential in using this type of delivery system in combination with traditional antibiotics to slow the development of resistance. Collectively, this study shows the promise of using a chloride-inducible promoter for the bacterial delivery of AMPs in the body for the treatment of vancomycin-resistant enterococci (VRE) and other antibiotic-resistant bacteria. PMID:25841002

  16. Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium.

    PubMed

    Geldart, Kathryn; Borrero, Juan; Kaznessis, Yiannis N

    2015-06-01

    Antibiotic-resistant enterococcal infections are a major concern in hospitals where patients with compromised immunity are readily infected. Enterococcus faecium bacteria are of particular interest as these pathogens account for over 80% of vancomycin-resistant enterococcal infections. Antimicrobial peptides (AMPs) produced at the site of infection by engineered bacteria may offer a potential alternative to traditional antibiotics for the treatment of resistant bacteria such as E. faecium. For this mode of delivery to be effective, it is essential to identify a suitable protein expression system that can be used in the desired delivery bacterium. In this study, we describe a promising chloride-inducible promoter and its application in the bacterial delivery of AMPs from Lactococcus lactis to reduce counts of E. faecium bacteria in vitro. Reporter gene studies show that at chloride concentrations found within the human intestines, the chloride-inducible promoter exhibits high levels of protein expression compared to those of the commonly used nisin-inducible promoter. These results indicate that this system is powerful and would not require the exogenous administration of an inducer molecule. In its application for AMP production against E. faecium in vitro, L. lactis producing AMPs under the chloride promoter rapidly decreased E. faecium counts by nearly 10,000-fold. As an extension of this application, we also demonstrate the potential in using this type of delivery system in combination with traditional antibiotics to slow the development of resistance. Collectively, this study shows the promise of using a chloride-inducible promoter for the bacterial delivery of AMPs in the body for the treatment of vancomycin-resistant enterococci (VRE) and other antibiotic-resistant bacteria. PMID:25841002

  17. Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

    PubMed

    Zhou, Qi-Jia; Wang, Jun; Liu, Min; Qiao, Ying; Hong, Wan-Shu; Su, Yong-Quan; Han, Kun-Huang; Ke, Qiao-Zhen; Zheng, Wei-Qiang

    2016-08-01

    As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion. PMID:27238427

  18. Regulation of hypothalamic neuropeptides gene expression in diet induced obesity resistant rats: possible targets for obesity prediction?

    PubMed

    Cifani, Carlo; Micioni Di Bonaventura, Maria V; Pucci, Mariangela; Giusepponi, Maria E; Romano, Adele; Di Francesco, Andrea; Maccarrone, Mauro; D'Addario, Claudio

    2015-01-01

    Several factors play a role in obesity (i.e., behavior, environment, and genetics) and epigenetic regulation of gene expression has emerged as a potential contributor in the susceptibility and development of obesity. To investigate the individual sensitivity to weight gain/resistance, we here studied gene transcription regulation of several hypothalamic neuropeptides involved in the control of energy balance in rats developing obesity (diet-induced obesity, DIO) or not (diet resistant, DR), when fed with a high fat diet. Rats have been followed up to 21 weeks of high fat diet exposure. After 5 weeks high fat diet exposure, the obese phenotype was developed and we observed a selective down-regulation of the orexigenic neuropeptide Y (NPY) and peroxisome proliferator-activated receptor gamma (PPAR-γ) genes. No changes were observed in the expression of the agouti-related protein (AgRP), as well as for all the anorexigenic genes under study. After long-term high fat diet exposure (21 weeks), NPY and PPAR-γ, as well as most of the genes under study, resulted not be different between DIO and DR, whereas a lower expression of the anorexigenic pro-opio-melanocortin (POMC) gene was observed in DIO rats when compared to DR rats. Moreover we observed that changes in NPY and POMC mRNA were inversely correlated with gene promoters DNA methylation. Our findings suggest that selective alterations in hypothalamic peptide genes regulation could contribute to the development of overweight in rats and that environmental factor, as in this animal model, might be partially responsible of these changes via epigenetic mechanism.

  19. Regulation of hypothalamic neuropeptides gene expression in diet induced obesity resistant rats: possible targets for obesity prediction?

    PubMed Central

    Cifani, Carlo; Micioni Di Bonaventura, Maria V.; Pucci, Mariangela; Giusepponi, Maria E.; Romano, Adele; Di Francesco, Andrea; Maccarrone, Mauro; D'Addario, Claudio

    2015-01-01

    Several factors play a role in obesity (i.e., behavior, environment, and genetics) and epigenetic regulation of gene expression has emerged as a potential contributor in the susceptibility and development of obesity. To investigate the individual sensitivity to weight gain/resistance, we here studied gene transcription regulation of several hypothalamic neuropeptides involved in the control of energy balance in rats developing obesity (diet-induced obesity, DIO) or not (diet resistant, DR), when fed with a high fat diet. Rats have been followed up to 21 weeks of high fat diet exposure. After 5 weeks high fat diet exposure, the obese phenotype was developed and we observed a selective down-regulation of the orexigenic neuropeptide Y (NPY) and peroxisome proliferator-activated receptor gamma (PPAR-γ) genes. No changes were observed in the expression of the agouti-related protein (AgRP), as well as for all the anorexigenic genes under study. After long-term high fat diet exposure (21 weeks), NPY and PPAR-γ, as well as most of the genes under study, resulted not be different between DIO and DR, whereas a lower expression of the anorexigenic pro-opio-melanocortin (POMC) gene was observed in DIO rats when compared to DR rats. Moreover we observed that changes in NPY and POMC mRNA were inversely correlated with gene promoters DNA methylation. Our findings suggest that selective alterations in hypothalamic peptide genes regulation could contribute to the development of overweight in rats and that environmental factor, as in this animal model, might be partially responsible of these changes via epigenetic mechanism. PMID:26106286

  20. The expression of analgesic-antitumor peptide (AGAP) from Chinese Buthus martensii Karsch in transgenic tobacco and tomato.

    PubMed

    Lai, Linlin; Huang, Tingting; Wang, Yi; Liu, Yanfeng; Zhang, Jinghai; Song, Yongbo

    2009-05-01

    The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze-thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally, transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study suggests a potential industrial application of AGAP expressed in plants.

  1. Septal Glucagon-Like Peptide 1 Receptor Expression Determines Suppression of Cocaine-Induced Behavior

    PubMed Central

    Harasta, Anne E; Power, John M; von Jonquieres, Georg; Karl, Tim; Drucker, Daniel J; Housley, Gary D; Schneider, Miriam; Klugmann, Matthias

    2015-01-01

    Glucagon-like peptide 1 (GLP-1) and its receptor GLP-1R are a key component of the satiety signaling system, and long-acting GLP-1 analogs have been approved for the treatment of type-2 diabetes mellitus. Previous reports demonstrate that GLP-1 regulates glucose homeostasis alongside the rewarding effects of food. Both palatable food and illicit drugs activate brain reward circuitries, and pharmacological studies suggest that central nervous system GLP-1 signaling holds potential for the treatment of addiction. However, the role of endogenous GLP-1 in the attenuation of reward-oriented behavior, and the essential domains of the mesolimbic system mediating these beneficial effects, are largely unknown. We hypothesized that the central regions of highest Glp-1r gene activity are essential in mediating responses to drugs of abuse. Here, we show that Glp-1r-deficient (Glp-1r−/−) mice have greatly augmented cocaine-induced locomotor responses and enhanced conditional place preference compared with wild-type (Glp-1r+/+) controls. Employing mRNA in situ hybridization we located peak Glp-1r mRNA expression in GABAergic neurons of the dorsal lateral septum, an anatomical site with a crucial function in reward perception. Whole-cell patch-clamp recordings of dorsal lateral septum neurons revealed that genetic Glp-1r ablation leads to increased excitability of these cells. Viral vector-mediated Glp-1r gene delivery to the dorsal lateral septum of Glp-1r−/− animals reduced cocaine-induced locomotion and conditional place preference to wild-type levels. This site-specific genetic complementation did not affect the anxiogenic phenotype observed in Glp-1r−/− controls. These data reveal a novel role of GLP-1R in dorsal lateral septum function driving behavioral responses to cocaine. PMID:25669605

  2. Identification of the thiazolyl peptide GE37468 gene cluster from Streptomyces ATCC 55365 and heterologous expression in Streptomyces lividans.

    PubMed

    Young, Travis S; Walsh, Christopher T

    2011-08-01

    Thiazolyl peptides are bacterial secondary metabolites that potently inhibit protein synthesis in Gram-positive bacteria and malarial parasites. Recently, our laboratory and others reported that this class of trithiazolyl pyridine-containing natural products is derived from ribosomally synthesized preproteins that undergo a cascade of posttranslational modifications to produce architecturally complex macrocyclic scaffolds. Here, we report the gene cluster responsible for production of the elongation factor Tu (EF-Tu)-targeting 29-member thiazolyl peptide GE37468 from Streptomyces ATCC 55365 and its heterologous expression in the model host Streptomyces lividans. GE37468 harbors an unusual β-methyl-δ-hydroxy-proline residue that may increase conformational rigidity of the macrocycle and impart reduced entropic costs of target binding. Isotope feeding and gene knockout were employed in the engineered S. lividans strain to identify the P450 monooxygenase GetJ as the enzyme involved in posttranslational transformation of isoleucine 8 to β-methyl-δ-hydroxy-proline through a predicted tandem double hydroxylation/cyclization mechanism. Loss of Ile8 oxygenative cyclization or mutation of Ile8 to alanine via preprotein gene replacement resulted in a 4-fold and 2-fold drop in antibiotic activity, respectively. This report of genetic manipulation of a 29-member thiazolyl peptide sets the stage for further genetic examination of structure activity relationships in the EF-Tu targeting class of thiazolyl peptides.

  3. Mutational analysis of the N-terminus in Schistocerca gregaria ion-transport peptide expressed in Drosophila Kc1 cells.

    PubMed

    Zhao, Y; Meredith, J; Brock, H W; Phillips, J E

    2005-01-01

    The functions of the 6-7 amino acid N-terminal domain conserved in insect and crustacean members of the hyperglycemic hormone (CHH) family were assayed by site-directed mutagenesis of Schistocerca gregaria ion-transport peptide (SchgrITP). Mutant peptides were expressed in Drosophila Kc1 cells and tested in a biological assay measuring stimulation of active Cl(-) transport across the locust ileum. We exchanged the N-terminal domain of SchgrITP with that of the shrimp Penaeus japonicus hyperglycemic hormone leaving the remainder of SchgrITP intact. The chimeric peptide was completely inactive in the ileal bioassay, showing that the N-terminus of SchgrITP is essential and that the 2 amino acids (phenylalanine-3 and aspartate-4) conserved in the shrimp and locust peptides are not sufficient for function. We made all possible alanine substitutions in the SchgrITP N-terminal domain. Only phenylalanines 2 and 3 were essential for function in the locust ileal bioassay. All N-terminal mutations were cleaved correctly from the prepropeptide, and expressed in similar concentrations as wild-type ITP suggesting the specific amino acids are not essential for these functions. Post-translational modification may explain a minor ITP isomorph observed in Drosophila Kc1 cell expression. Alanine substitution at position 2 produced a weak ITP antagonist. These structure-function studies, the first for any member of the CHH family, show that both conserved and unconserved amino acids contribute to SchgrITP ion-transport function and that the conserved aspartate in position 4 is required for a yet uncharacterized function.

  4. The expression and phylogenetics of the Inhibitor Cysteine Knot peptide OCLP1 in the honey bee Apis mellifera.

    PubMed

    Bloch, Guy; Cohen, Mira

    2014-06-01

    Small cysteine-rich peptides have diverse functions in insects including antimicrobial defense, phenoloxidase activity regulation, and toxic inhibition of ion channels of prey or predator. We combined bioinformatics and measurements of transcript abundance to start characterizing AmOCLP1, a recently discovered Inhibitor Cysteine Knot peptide in the honey bee Apis mellifera. We found that the genomes of ants, bees, and the wasp Nasonia vitripennis encode orthologous sequences indicating that OCLP1 is a conserved peptide and not unique to the honey bee. Search of available EST libraries and quantitative real time PCR analyses indicate that the transcript of AmOCLP1 is ubiquitous with expression in life stages ranging from embryos to adults and in all tested tissues. In worker honey bees AmOCLP1 expression was not associated with age or task and did not show clear enrichment in any of the tested tissues. There was however a consistent trend toward higher transcript levels in the abdomen of foragers relative to levels in the head or thorax, and compared to levels in the abdomen of younger worker bees. By contrast, in drones AmOCLP1 transcript levels appeared higher in the head relative to the abdomen. Finer analyses of the head and abdomen indicated that the AmOCLP1 transcript is not enriched in the stinger and the associated venom sac or in cephalic exocrine glands. The evolutionary conservation in the Hymenoptera, the ubiquitous expression, and the lack of enrichment in the venom gland, stinger, exocrine glands, and the brain are not consistent with the hypotheses that OCLP1 is a secreted honeybee toxin or an endotoxin acting in the central nervous system. Rather we hypothesize that OCLP1 is a conserved antimicrobial or phenoloxidase inhibitor peptide.

  5. The expression and phylogenetics of the Inhibitor Cysteine Knot peptide OCLP1 in the honey bee Apis mellifera.

    PubMed

    Bloch, Guy; Cohen, Mira

    2014-06-01

    Small cysteine-rich peptides have diverse functions in insects including antimicrobial defense, phenoloxidase activity regulation, and toxic inhibition of ion channels of prey or predator. We combined bioinformatics and measurements of transcript abundance to start characterizing AmOCLP1, a recently discovered Inhibitor Cysteine Knot peptide in the honey bee Apis mellifera. We found that the genomes of ants, bees, and the wasp Nasonia vitripennis encode orthologous sequences indicating that OCLP1 is a conserved peptide and not unique to the honey bee. Search of available EST libraries and quantitative real time PCR analyses indicate that the transcript of AmOCLP1 is ubiquitous with expression in life stages ranging from embryos to adults and in all tested tissues. In worker honey bees AmOCLP1 expression was not associated with age or task and did not show clear enrichment in any of the tested tissues. There was however a consistent trend toward higher transcript levels in the abdomen of foragers relative to levels in the head or thorax, and compared to levels in the abdomen of younger worker bees. By contrast, in drones AmOCLP1 transcript levels appeared higher in the head relative to the abdomen. Finer analyses of the head and abdomen indicated that the AmOCLP1 transcript is not enriched in the stinger and the associated venom sac or in cephalic exocrine glands. The evolutionary conservation in the Hymenoptera, the ubiquitous expression, and the lack of enrichment in the venom gland, stinger, exocrine glands, and the brain are not consistent with the hypotheses that OCLP1 is a secreted honeybee toxin or an endotoxin acting in the central nervous system. Rather we hypothesize that OCLP1 is a conserved antimicrobial or phenoloxidase inhibitor peptide. PMID:24721445

  6. Large-scale production of soluble recombinant amyloid-β peptide 1-42 using cold-inducible expression system.

    PubMed

    Kim, Eun-Kyung; Moon, Jeong Chan; Lee, Jeong Mi; Jeong, Min Seop; Oh, Choongseob; Ahn, Sung-Min; Yoo, Yung Joon; Jang, Ho Hee

    2012-11-01

    Amyloid-β peptide 1-42 (Aβ(1-42)), the predominant form in senile plaques, plays important roles in the pathogenesis of Alzheimer's disease. Because Aβ(1-42) has aggregation-prone nature, it has been difficult to produce in a soluble state in bacterial expression systems. In this study, we modified our expression system to increase the soluble fraction of Aβ(1-42) in Escherichia coli (E. coli) cells. The expression level and solubility of recombinant Aβ(1-42) induced at the low temperature (16°C) is highly increased compared to that induced at 37°C. To optimize expression temperature, the coding region of Aβ(1-42) was constructed in a pCold vector, pCold-TF, which has a hexahistidine-tagged trigger factor (TF). Recombinant Aβ(1-42) was expressed primarily as a soluble protein using pCold vector system and purified with a nickel-chelating resin. When the toxic effect of recombinant Aβ(1-42) examined on human neuroblastoma SH-SY5Y cells, the purified Aβ(1-42) induced cell toxicity on SH-SY5Y cells. In conclusion, the system developed in this study will provide a useful method for the production of aggregation prone-peptide such as Aβ(1-42).

  7. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids

    PubMed Central

    Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J.; Wilkinson, Trevor C. I.

    2016-01-01

    The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these “undesirable” residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

  8. Expression analysis and identification of antimicrobial peptide transcripts from six North American frog species

    USGS Publications Warehouse

    Robertson, Laura S.; Fellers, Gary M.; Marranca, Jamie Marie; Kleeman, Patrick M.

    2013-01-01

    Frogs secrete antimicrobial peptides onto their skin. We describe an assay to preserve and analyze antimicrobial peptide transcripts from field-collected skin secretions that will complement existing methods for peptide analysis. We collected skin secretions from 4 North American species in the field in California and 2 species in the laboratory. Most frogs appeared healthy after release; however, Rana boylii in the Sierra Nevada foothills, but not the Coast Range, showed signs of morbidity and 2 died after handling. The amount of total RNA extracted from skin secretions was higher in R. boylii and R. sierrae compared to R. draytonii, and much higher compared to Pseudacris regilla. Interspecies variation in amount of RNA extracted was not explained by size, but for P. regilla it depended upon collection site and date. RNA extracted from skin secretions from frogs handled with bare hands had poor quality compared to frogs handled with gloves or plastic bags. Thirty-four putative antimicrobial peptide precursor transcripts were identified. This study demonstrates that RNA extracted from skin secretions collected in the field is of high quality suitable for use in sequencing or quantitative PCR (qPCR). However, some species do not secrete profusely, resulting in very little extracted RNA. The ability to measure transcript abundance of antimicrobial peptides in field-collected skin secretions complements proteomic analyses and may provide insight into transcriptional mechanisms that could affect peptide abundance.

  9. Molecular cloning and expression analysis of liver-expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 genes in the blunt snout bream (Megalobrama amblycephala).

    PubMed

    Liang, Tao; Ji, Wei; Zhang, Gui-Rong; Wei, Kai-Jian; Feng, Ke; Wang, Wei-Min; Zou, Gui-Wei

    2013-08-01

    Liver-expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 are widespread in fish and extremely important components of the host innate immune system. In this study, full-length cDNAs of LEAP-1 and LEAP-2 were cloned and sequenced from blunt snout bream, Megalobrama amblycephala. The open reading frames (ORF) of LEAP-1 and LEAP-2 genes encode putative peptides of 94 and 92 amino acids, which possess eight and four conserved cysteine residues, respectively. The homologous identities of deduced amino acid sequences show that the LEAP-1 and LEAP-2 of blunt snout bream share considerable similarity with those of grass carp. The mRNA expressions of LEAP-1 and LEAP-2 were detectable at different early developmental stages of blunt snout bream and varied with embryonic and larval growth. LEAP-1 and LEAP-2 were expressed in a wide range of adult tissues, with the highest expression levels in the liver and midgut, respectively. Bacterial challenge experiments showed that the levels of LEAP-1 and LEAP-2 mRNA expression were up-regulated in the liver, spleen, gill and brain of juvenile blunt snout bream. These results indicate that the LEAP-1 and LEAP-2 may play important roles in early development of embryos and fry, and may contribute to the defense against the pathogenic bacterial invasion. This study will further our understanding of the function of LEAP-1 and LEAP-2 and the molecular mechanism of innate immunity in teleosts.

  10. Impact of glucose infusion on the structural and functional characteristics of adipose tissue and on hypothalamic gene expression for appetite regulatory neuropeptides in the sheep fetus during late gestation

    PubMed Central

    Mühlhäusler, BS; Adam, CL; Marrocco, EM; Findlay, PA; Roberts, CT; McFarlane, JR; Kauter, KG; McMillen, IC

    2005-01-01

    In the present study, our aim was to determine whether intrafetal glucose infusion increases fetal adiposity, synthesis and secretion of leptin and regulates gene expression of the ‘appetite regulatory’ neuropeptides neuropepetide Y (NPY), agouti-related peptide (AGRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) and receptors (leptin receptor (OB-Rb) and melancortin 3 receptor (MC3R)) within the fetal hypothalamus. Glucose (50% dextrose in saline) or saline was infused (7.5 ml h−1) into fetal sheep between 130 and 140 days gestation (term = 150 ± 3 days gestation). Glucose infusion increased circulating glucose and insulin concentrations, mean lipid locule size (532.8 ± 3.3 μm2 versus 456.7 ± 14.8 μm2) and total unilocular fat mass (11.7 ± 0.6 g versus 8.9 ± 0.6 g) of the perirenal fat depot. The expression of OB-Rb mRNA was higher in the ventromedial nucleus compared to the arcuate nucleus of the hypothalamus in both glucose and saline infused fetuses (F= 8.04; P < 0.01) and there was a positive correlation between expression of OB-Rb and MC3R mRNA in the arcuate nucleus (r= 0.81; P < 0.005). Glucose infusion increased mRNA expression for POMC, but not for the anorectic neuropeptide CART, or the orexigenic neuropeptides NPY and AGRP, in the arcuate nucleus of the fetal hypothalamus. These findings demonstrate that increased circulating glucose and insulin regulate gene expression of the neuropeptides within the fetal hypothalamus that are part of the neural network regulating energy balance in adult life. PMID:15661821

  11. Identification, expression, and innate immune responses of two insulin-like peptide genes in the razor clam Sinonovacula constricta.

    PubMed

    Niu, Donghong; Wang, Fei; Zhao, Honggang; Wang, Ze; Xie, Shumei; Li, Jiale

    2016-04-01

    Insulin-like peptide (ILP) has emerged as a cell regulatory factor with multiple functions in vertebrates and invertebrates. In the present study, we identified and characterized two ILP genes, ILP1 and ILP2, in the razor clam Sinonovacula constricta. Both ILPs have a signal peptide and a mature domain consisting of six strictly conserved cysteines. The tertiary structure is divided into three main α-helices with a C-domain loop that separates helix 1 from helix 2. Both of ILPs were found to be regulated according to tissue type and developmental stage. After challenge with Vibrio anguillarum, Vibrio parahaemolyticus and Micrococcus lysodeikticus, the expression of two ILP genes was significantly up-regulated in the liver, hemocytes and mantle tissues, suggesting that the ILPs may play roles in the innate immunity in the razor clam Sinonovacula constricta. PMID:26980611

  12. Design, characterization and expression of a novel hybrid peptides melittin (1-13)-LL37 (17-30).

    PubMed

    Wu, Rujuan; Wang, Qing; Zheng, Zhaojun; Zhao, Longmei; Shang, Yajing; Wei, Xubiao; Liao, Xiudong; Zhang, Rijun

    2014-07-01

    Hybridizing of different antimicrobial peptides (AMPs) has been a common practice for obtaining novel hybrid AMPs with elevated antibacterial activity but minimized cytotoxicity. The hybrid peptides melittin (1-13)-LL37 (17-30) (M-L) combining the hydrophobic N-terminal fragment of melittin (M) with the core antibacterial fragment of LL37 (L), was designed for the first time to explore its antibacterial activity and hemolytic activity against bacteria and sheep erythrocyte respectively. Results showed that M-L had an even more potent antibacterial activity against all indicator strains (especially gram-positive bacteria) than M and L, whereas didn't exhibit hemolytic activity to sheep erythrocytes, implying M-L can be served as a potential therapeutic drug to substitute traditional antibiotics. However the high expense of biosynthesis limited its further research, therefore fusion expression of M-L was carried out in Escherichia coli (E. coli) for overproducing the hybrid peptide so as to solve the problem. The DNA sequence encoding M-L with preferred codons was cloned into the pET-SUMO vector for protein expression in E. coli BL21 (DE3). After IPTG induction, approximately 165 mg soluble fusion protein SUMO-M-L was recovered per liter supernatant of the fermentation ultrasonic lysate using Ni-NTA Sepharose column (92 % purity). And 23 mg recombinant M-L was obtained per liter culture after cleavage of SUMO protease and purification of Ni-NTA Sepharose column. In sum, this research not only supplied an effective approach for overproducing hybrid peptide M-L, but paved the way for its further exploration on pharmaceutical potential and medical importance. PMID:24871991

  13. Design, characterization and expression of a novel hybrid peptides melittin (1-13)-LL37 (17-30).

    PubMed

    Wu, Rujuan; Wang, Qing; Zheng, Zhaojun; Zhao, Longmei; Shang, Yajing; Wei, Xubiao; Liao, Xiudong; Zhang, Rijun

    2014-07-01

    Hybridizing of different antimicrobial peptides (AMPs) has been a common practice for obtaining novel hybrid AMPs with elevated antibacterial activity but minimized cytotoxicity. The hybrid peptides melittin (1-13)-LL37 (17-30) (M-L) combining the hydrophobic N-terminal fragment of melittin (M) with the core antibacterial fragment of LL37 (L), was designed for the first time to explore its antibacterial activity and hemolytic activity against bacteria and sheep erythrocyte respectively. Results showed that M-L had an even more potent antibacterial activity against all indicator strains (especially gram-positive bacteria) than M and L, whereas didn't exhibit hemolytic activity to sheep erythrocytes, implying M-L can be served as a potential therapeutic drug to substitute traditional antibiotics. However the high expense of biosynthesis limited its further research, therefore fusion expression of M-L was carried out in Escherichia coli (E. coli) for overproducing the hybrid peptide so as to solve the problem. The DNA sequence encoding M-L with preferred codons was cloned into the pET-SUMO vector for protein expression in E. coli BL21 (DE3). After IPTG induction, approximately 165 mg soluble fusion protein SUMO-M-L was recovered per liter supernatant of the fermentation ultrasonic lysate using Ni-NTA Sepharose column (92 % purity). And 23 mg recombinant M-L was obtained per liter culture after cleavage of SUMO protease and purification of Ni-NTA Sepharose column. In sum, this research not only supplied an effective approach for overproducing hybrid peptide M-L, but paved the way for its further exploration on pharmaceutical potential and medical importance.

  14. Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

    PubMed Central

    Boll, M; Herget, M; Wagener, M; Weber, W M; Markovich, D; Biber, J; Clauss, W; Murer, H; Daniel, H

    1996-01-01

    The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences. Images Fig. 7 PMID:8552623

  15. Neuropeptide S receptor 1 expression in the intestine and skin--putative role in peptide hormone secretion.

    PubMed

    Sundman, L; Saarialho-Kere, U; Vendelin, J; Lindfors, K; Assadi, G; Kaukinen, K; Westerholm-Ormio, M; Savilahti, E; Mäki, M; Alenius, H; D'Amato, M; Pulkkinen, V; Kere, J; Saavalainen, P

    2010-01-01

    Neuropeptide S receptor 1 (NPSR1) was recently found to be genetically associated with inflammatory bowel disease in addition to asthma and related traits. Epithelia of several organs express NPSR1 isoforms A and B, including the intestine and the skin, and NPSR1 appears to be upregulated in inflammation. In this study, we used cell lines and tissue samples to characterize the expression of NPSR1 and its ligand neuropeptide S (NPS) in inflammation. We used polyclonal and monoclonal antibodies to investigate the expression of NPS and NPSR1 in intestinal diseases, such as celiac disease and food allergy, and in cutaneous inflammatory disorders. We found that NPSR1-A was expressed by the enteroendocrine cells of the gut. Overall, the expression pattern of NPS was similar to its receptor suggesting an autocrine mechanism. In an NPSR1-A overexpressing cell model, stimulation with NPS resulted in a dose-dependent upregulation of glycoprotein hormone, alpha polypeptide (CGA), tachykinin 1 (TAC1), neurotensin (NTS) and galanin (GAL) encoding peptide hormones secreted by enteroendocrine cells. Because NPSR1 was also expressed in macrophages, neutrophils, and intraepithelial lymphocytes, we demonstrated that stimulation with the pro-inflammatory cytokines tumour necrosis factor alpha and interferon gamma increased NPSR1 expression in the THP-1 monocytic cells. In conclusion, similar to other neuropeptides and their receptors, NPSR1 signalling might play a dual role along the gut-brain axis. The NPS/NPSR1 pathway may participate in the regulation of the peptide hormone production in enteroendocrine cells of the small intestine.

  16. Cocaine-and Amphetamine Regulated Transcript (CART) Peptide Is Expressed in Precursor Cells and Somatotropes of the Mouse Pituitary Gland

    PubMed Central

    Mortensen, Amanda H.

    2016-01-01

    Cocaine-and Amphetamine Regulated Transcript (CART) peptide is expressed in the brain, endocrine and neuroendocrine systems and secreted into the serum. It is thought to play a role in regulation of hypothalamic pituitary functions. Here we report a spatial and temporal analysis of Cart expression in the pituitaries of adult and developing normal and mutant mice with hypopituitarism. We found that Prop1 is not necessary for initiation of Cart expression in the fetal pituitary at e14.5, but it is required indirectly for maintenance of Cart expression in the postnatal anterior pituitary gland. Pou1f1 deficiency has no effect on Cart expression before or after birth. There is no 1:1 correspondence between CART and any particular cell type. In neonates, CART is detected primarily in non-proliferating, POU1F1-positive cells. CART is also found in some cells that express TSH and GH suggesting a correspondence with committed progenitors of the POU1F1 lineage. In summary, we have characterized the normal temporal and cell specific expression of CART in mouse development and demonstrate that postnatal CART expression in the pituitary gland requires PROP1. PMID:27685990

  17. Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

    PubMed Central

    Ebina, Isao; Takemoto-Tsutsumi, Mariko; Watanabe, Shun; Koyama, Hiroaki; Endo, Yayoi; Kimata, Kaori; Igarashi, Takuya; Murakami, Karin; Kudo, Rin; Ohsumi, Arisa; Noh, Abdul Latif; Takahashi, Hiro; Naito, Satoshi; Onouchi, Hitoshi

    2015-01-01

    Upstream open reading frames (uORFs) are often found in the 5′-leader regions of eukaryotic mRNAs and can negatively modulate the translational efficiency of the downstream main ORF. Although the effects of most uORFs are thought to be independent of their encoded peptide sequences, certain uORFs control translation of the main ORF in a peptide sequence-dependent manner. For genome-wide identification of such peptide sequence-dependent regulatory uORFs, exhaustive searches for uORFs with conserved amino acid sequences have been conducted using bioinformatic analyses. However, whether the conserved uORFs identified by these bioinformatic approaches encode regulatory peptides has not been experimentally determined. Here we analyzed 16 recently identified Arabidopsis thaliana conserved uORFs for the effects of their amino acid sequences on the expression of the main ORF using a transient expression assay. We identified five novel uORFs that repress main ORF expression in a peptide sequence-dependent manner. Mutational analysis revealed that, in four of them, the C-terminal region of the uORF-encoded peptide is critical for the repression of main ORF expression. Intriguingly, we also identified one exceptional sequence-dependent regulatory uORF, in which the stop codon position is not conserved and the C-terminal region is not important for the repression of main ORF expression. PMID:25618853

  18. Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria.

    PubMed

    Wu, Tingquan; Tang, Dingzhong; Chen, Weida; Huang, Hexun; Wang, Rui; Chen, Yongfang

    2013-09-15

    Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed

  19. Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects.

    PubMed

    Kimura, Richard H; Cheng, Zhen; Gambhir, Sanjiv Sam; Cochran, Jennifer R

    2009-03-15

    There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity ( approximately 10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or (64)Cu-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC(50), approximately 20 nmol/L) (64)Cu-DOTA-conjugated knottin peptides was 4.47% +/- 1.21% and 4.56% +/- 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC(50), approximately 0.4 mumol/L; 1.48 +/- 0.53%ID/g) and c(RGDyK) (IC(50), approximately 1 mumol/L; 2.32 +/- 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, (64)Cu-DOTA-conjugated knottin peptides generated lower levels of nonspecific liver uptake ( approximately 2%ID/g) compared with c(RGDyK) ( approximately 4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. (64)Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers.

  20. Β-defensin in Nile tilapia (Oreochromis niloticus): Sequence, tissue expression, and anti-bacterial activity of synthetic peptides.

    PubMed

    Dong, Jun-Jian; Wu, Fang; Ye, Xing; Sun, Cheng-Fei; Tian, Yuan-Yuan; Lu, Mai-Xin; Zhang, Rui; Chen, Zhi-Hang

    2015-07-15

    Beta-defensins (β-defensins) are small cationic amphiphilic peptides that are widely distributed in plants, insects, and vertebrates, and are important for their antimicrobial properties. In this study, the β-defensin (Onβ-defensin) gene of the Nile tilapia (Oreochromis niloticus) was cloned from spleen tissue. Onβ-defensin has a genomic DNA sequence of 674 bp and produces a cDNA of 454 bp. Sequence alignments showed that Onβ-defensin contains three exons and two introns. Sequence analysis of the cDNA identified an open reading frame of 201 bp, encoding 66 amino acids. Bioinformatic analysis showed that Onβ-defensin encodes a cytoplasmic protein molecule containing a signal peptide. The deduced amino acid sequence of this peptide contains six conserved cysteine residues and two conserved glycine residues, and shows 81.82% and 78.33% sequence similarities with β-defensin-1 of fugu (Takifugu rubripes) and rainbow trout (Oncorhynchus mykiss), respectively. Real-time quantitative PCR showed that the level of Onβ-defensin expression was highest in the skin (307.1-fold), followed by the spleen (77.3-fold), kidney (17.8-fold), and muscle (16.5-fold) compared to controls. By contrast, low levels of expression were found in the liver, heart, intestine, stomach, and gill (<3.0-fold). Artificial infection of tilapia with Streptococcus agalactiae (group B streptococcus [GBS] strain) resulted in a significantly upregulated expression of Onβ-defensin in the skin, muscle, kidney, and gill. In vitro antimicrobial experiments showed that a synthetic Onβ-defensin polypeptide had a certain degree of inhibitory effect on the growth of Escherichia coli DH5α and S. agalactiae. The results indicate that Onβ-defensin plays a role in immune responses that suppress or kill pathogens.

  1. Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes.

    PubMed

    Ochoa-Zarzosa, Alejandra; Villarreal-Fernández, Edith; Cano-Camacho, Horacio; López-Meza, Joel E

    2009-07-01

    A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25-0.5mM) reduces approximately 50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), beta-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC. PMID:19393738

  2. Live detection and purification of cells based on the expression of a histone chaperone, HIRA, using a binding peptide.

    PubMed

    Kochurani, K J; Suganya, Annie A; Nair, Madhumathy G; Louis, Jiss Maria; Majumder, Aditi; Kumar, Santhosh K; Abraham, Parvin; Dutta, Debasree; Maliekal, Tessy T

    2015-01-01

    Flow cytometry is a reliable method for identification and purification of live cells from a heterogeneous population. Since permeabilized cells cannot be sorted live in a FACS sorter, its application in isolation of functional cells largely depends on antibodies for surface markers. In various fields of biology we find intracellular markers that reveal subpopulations of biological significance. Cell cycle stage specific molecules, metastatic signature molecules, stemness associated proteins etc. are examples of potential markers that could improve the research and therapy enormously. Currently their use is restricted by lack of techniques that allow live detection. Even though a few methods like aptamers, droplet-based microfluidics and smartflares are reported, their application is limited. Here, for the first time we report a simple, cost-effective and efficient method of live sorting of cells based on the expression of an intracellular marker using a fluorophore-tagged binding peptide. The target molecule selected was a histone chaperone, HIRA, the expression of which can predict the fate of differentiating myoblast. Our results confirm that the peptide shows specific interaction with its target; and it can be used to separate cells with differential expression of HIRA. Further, this method offers high purity and viability for the isolated cells. PMID:26596463

  3. Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1.

    PubMed

    Cao, Zhen; Wu, Xue-Fei; Peng, Yan; Zhang, Rui; Li, Na; Yang, Jin-Yi; Zhang, Shu-Qin; Zhang, Wan-Qin; Zhao, Jie; Li, Shao

    2015-11-01

    Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine.

  4. Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes.

    PubMed

    Ochoa-Zarzosa, Alejandra; Villarreal-Fernández, Edith; Cano-Camacho, Horacio; López-Meza, Joel E

    2009-07-01

    A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25-0.5mM) reduces approximately 50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), beta-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC.

  5. Limitations for purification of murine interleukin-18 when expressed as a fusion protein containing the FLAG peptide.

    PubMed

    Elhofy, A; Bost, K L

    1998-09-01

    As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system. The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affinity columns could be performed. While significant amounts of recombinant IL-18 were present in E. coli lysates, only a small portion of this material could be recovered on immunoaffinity columns conjugated with an anti-FLAG antibody. Surprisingly, the majority of recombinant IL-18 present in E. coli (strain JM83) bacterial lysates did not contain the FLAG peptide and therefore did not bind to immunoaffinity columns conjugated with an anti-FLAG antibody. However, we found that the BL21 strain of E. coli, which has reduced endogenous protease activity, could express the majority of recombinant IL-18 as the fusion protein, FLAG-IL-18. Taken together, these studies show that it is necessary to consider whether protease sites formed at the FLAG-protein junction can be easily cleaved by the bacterial strain used to express the fusion protein.

  6. Isolation, characterization, and expression analyses of plant elicitor peptides (pep) genes in maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PROPEP1, PROPEP 2, and PROPEP3 genes appear to have roles in a feedback loop that amplifies defense signaling pathways initiated by pathogens. We present evidence to support the role of peptides derived from PROPEP genes as endogenous elicitors that are generated in response to pathogens. The preval...

  7. Isolation, characterization, and expression analyses of plant elicitor peptides (Pep) genes in maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect-induced defenses occur in nearly all plants and are regulated by conserved signaling pathways. In plant families, peptides with analogous activity have remained elusive. Peps are conserved signals across diverse plant families regulating antiherbivore defenses and are likely to be the missing...

  8. Characterization and regulation of expression of an antifungal peptide from hemolymph of an insect, Manduca sexta.

    PubMed

    Al Souhail, Qasim; Hiromasa, Yasuaki; Rahnamaeian, Mohammad; Giraldo, Martha C; Takahashi, Daisuke; Valent, Barbara; Vilcinskas, Andreas; Kanost, Michael R

    2016-08-01

    Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC50 of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta. PMID:26976231

  9. Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

    PubMed

    Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

    2013-03-01

    Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.

  10. Capsule expression in Streptococcus mitis modulates interaction with oral keratinocytes and alters susceptibility to human antimicrobial peptides.

    PubMed

    Rukke, H V; Engen, S A; Schenck, K; Petersen, F C

    2016-08-01

    Streptococcus mitis is a colonizer of the oral cavity and the nasopharynx, and is closely related to Streptococcus pneumoniae. Both species occur in encapsulated and unencapsulated forms, but in S. mitis the role of the capsule in host interactions is mostly unknown. Therefore, the aim of this study was to examine how capsule expression in S. mitis can modulate interactions with the host with relevance for colonization. The S. mitis type strain, as well as two mutants of the type strain, an isogenic capsule deletion mutant, and a capsule switch mutant expressing the serotype 4 capsule of S. pneumoniae TIGR4, were used. Wild-type and capsule deletion strains of S. pneumoniae TIGR4 were included for comparison. We found that capsule production in S. mitis reduced adhesion to oral and lung epithelial cells. Further, exposure of oral epithelial cells to encapsulated S. mitis resulted in higher interleukin-6 and CXCL-8 transcription levels relative to the unencapsulated mutant. Capsule expression in S. mitis increased the sensitivity to human neutrophil peptide 1-3 but reduced the sensitivity to human β-defensin-3 and cathelicidin. This was in contrast with S. pneumoniae in which capsule expression has been generally associated with increased sensitivity to human antimicrobial peptides (AMPs). Collectively, these findings indicate that capsule expression in S. mitis is important in modulating interactions with epithelial cells, and is associated with increased or reduced susceptibility to AMPs depending on the nature of the AMP.

  11. Construction and expression of an antimicrobial peptide scolopin 1 from the centipede venoms of Scolopendra subspinipes mutilans in Escherichia coli using SUMO fusion partner.

    PubMed

    Hou, Huanhuan; Yan, Weili; Du, Kexing; Ye, Yangjing; Cao, Qianqian; Ren, Wenhua

    2013-12-01

    Antimicrobial peptide scolopin 1 (AMP-scolopin 1) is a small cationic peptide identified from centipede venoms of Scolopendra subspinipes mutilans. It has broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We first report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antimicrobial peptide AMP-scolopin 1. The fusion protein expressed in a soluble form was purified to a purity of 95% by Ni-IDA chromatography. After the SUMO-scolopin 1 fusion protein was cleaved by the SUMO protease at 30°C for 1 h, the cleaved sample was reapplied to a Ni-IDA. The recombinant scolopin1 had similar antimicrobial properties to the synthetic scolopin 1. Thus, we successfully established a system for purifying peptide of centipede, which could be used for further research.

  12. Construction and expression of an antimicrobial peptide scolopin 1 from the centipede venoms of Scolopendra subspinipes mutilans in Escherichia coli using SUMO fusion partner.

    PubMed

    Hou, Huanhuan; Yan, Weili; Du, Kexing; Ye, Yangjing; Cao, Qianqian; Ren, Wenhua

    2013-12-01

    Antimicrobial peptide scolopin 1 (AMP-scolopin 1) is a small cationic peptide identified from centipede venoms of Scolopendra subspinipes mutilans. It has broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We first report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antimicrobial peptide AMP-scolopin 1. The fusion protein expressed in a soluble form was purified to a purity of 95% by Ni-IDA chromatography. After the SUMO-scolopin 1 fusion protein was cleaved by the SUMO protease at 30°C for 1 h, the cleaved sample was reapplied to a Ni-IDA. The recombinant scolopin1 had similar antimicrobial properties to the synthetic scolopin 1. Thus, we successfully established a system for purifying peptide of centipede, which could be used for further research. PMID:24145284

  13. Expression pattern of sonic hedgehog signaling and calcitonin gene-related peptide in the socket healing process after tooth extraction.

    PubMed

    Pang, Pai; Shimo, Tsuyoshi; Takada, Hiroyuki; Matsumoto, Kenichi; Yoshioka, Norie; Ibaragi, Soichiro; Sasaki, Akira

    2015-11-01

    Sonic Hedgehog (SHH), a neural development inducer, plays a significant role in the bone healing process. Calcitonin gene-related peptide (CGRP), a neuropeptide marker of sensory nerves, has been demonstrated to affect bone formation. The roles of SHH signaling and CGRP-positive sensory nerves in the alveolar bone formation process have been unknown. Here we examined the expression patterns of SHH signaling and CGRP in mouse socket by immunohistochemistry and immunofluorescence analysis. We found that the expression level of SHH peaked at day 3 and was then decreased at 5 days after tooth extraction. CGRP, PTCH1 and GLI2 were each expressed in a similar pattern with their highest expression levels at day 5 and day 7 after tooth extraction. CGRP and GLI2 were co-expressed in some inflammatory cells and bone forming cells. In some areas, CGRP-positive neurons expressed GLI2. In conclusion, SHH may affect alveolar bone healing by interacting with CGRP-positive sensory neurons and thus regulate the socket's healing process after tooth extraction. PMID:26427874

  14. Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells

    PubMed Central

    Chin, Joanna Y; Reza, Faisal; Glazer, Peter M

    2013-01-01

    Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes. PMID:23337982

  15. The expression of leptin, hypothalamic neuropeptides and UCP1 before, during and after fattening in the Daurian ground squirrel (Spermophilus dauricus).

    PubMed

    Xing, Xin; Yang, Ming; Wang, De-Hua

    2015-06-01

    The Daurian ground squirrel (Spermophilus dauricus) accumulates large amounts of body fat during pre-hibernation fattening. Leptin, an adipose-derived hormone, plays important roles in energy balance and thermogenesis. We predicted that body fat accumulation would lead to the elevation of leptin concentration while its effect on satiety would be suppressed in hypothalamus during fattening. In addition, the uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) would increase and correlated positively with leptin concentration before hibernation. Here, we measured serum leptin concentration and leptin mRNA in white adipose tissue (WAT), hypothalamic neuropeptides involved in energy regulation and UCP1 in BAT before, during and after fattening in squirrels. The fat mass gradually increased during fattening but serum leptin increased mainly in the late phase of fattening, which was consistent with leptin mRNA expression in WAT. During fattening, the mRNA of hypothalamic leptin receptor was up-regulated and correlated positively with serum leptin. Orexigenic neuropeptide Y mRNA increased by 67%; however agouti-related peptide remained unchanged before hibernation. There was no significant change in anorexigenic neuropeptide mRNA. No change in suppressor of cytokine signaling-3 and protein tyrosine phosphatase-1B was detected. UCP1 mRNA expression and protein content in BAT increased significantly after fattening. These changes were independent of environmental conditions and serum leptin concentration. Our results suggest that the dissociation of leptin production and adiposity during fattening may facilitate fat accumulation. No evidence of suppressed leptin signal was found in fattening squirrels. The UCP1 recruitment in post-fattening squirrels could occur without winter-like acclimation and increased leptin.

  16. Early weaning is associated with higher neuropeptide Y (NPY) and lower cocaine- and amphetamine-regulated transcript (CART) expressions in the paraventricular nucleus (PVN) in adulthood.

    PubMed

    Younes-Rapozo, Viviane; de Moura, Egberto Gaspar; da Silva Lima, Natália; Barradas, Penha Cristina; Manhães, Alex C; de Oliveira, Elaine; Lisboa, Patricia Cristina

    2012-12-28

    The interruption of lactation for a short period, without the use of pharmacological substances or maternal separation, causes offspring malnutrition and hypoleptinaemia and programmes for metabolic disorders such as higher body weight and adiposity, hyperphagia, hyperleptinaemia and central leptin resistance in adulthood. Here, in order to clarify the mechanisms underlying the phenotype observed in adult early-weaned (EW) rats, we studied the expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in different hypothalamic nuclei by immunohistochemistry and Western blot. In the EW group, the teats of lactating rats were blocked with a bandage to interrupt lactation during the last 3 d, while control pups had free access to milk throughout the entire lactation period. At age 180 d, EW offspring showed higher NPY staining in the paraventricular nucleus (PVN), as well as NPY protein content (+68 %) in total hypothalamus than control ones. AgRP showed no changes in staining or Western blot. POMC content was not affected; however, its distribution pattern was altered. CART-positive cells of EW offspring had lower immunoreactivity associated with reduced cell number in the PVN and lower protein content ( - 38 %) in total hypothalamus. The present data indicate that precocious weaning can imprint the neuronal circuitry, especially in the PVN, and cause a long-term effect on the expression of specific orexigenic and anorexigenic neuropeptides, such as NPY and CART, that can be caused by leptin resistance and are coherent with the hyperphagia observed in these animals.

  17. C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression

    NASA Astrophysics Data System (ADS)

    Scotland, Ramona S.; Cohen, Marc; Foster, Paul; Lovell, Matthew; Mathur, Anthony; Ahluwalia, Amrita; Hobbs, Adrian J.

    2005-10-01

    The multifaceted process of immune cell recruitment to sites of tissue injury is key to the development of an inflammatory response and involved in the pathogenesis of numerous cardiovascular disorders. We recently identified C-type natriuretic peptide (CNP) as an important endothelium-derived mediator that regulates vascular tone and protects against myocardial ischemia/reperfusion injury. Herein, we investigated whether CNP inhibits leukocyte recruitment and platelet aggregation and thereby exerts a potential antiinflammatory influence on the blood vessel wall. We assessed the effects of CNP on leukocyte-endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in animals with high basal leukocyte activation (endothelial nitric oxide synthase knockout mice, eNOS-/-) or under acute inflammatory conditions (induced by interleukin-1 or histamine). CNP suppressed basal leukocyte rolling in eNOS-/- mice in a rapid, reversible, and concentration-dependent manner. These effects of CNP were mimicked by the selective natriuretic peptide receptor-C agonist cANF4-23. CNP also suppressed leukocyte rolling induced by IL-1 or histamine, inhibited platelet-leukocyte interactions, and prevented thrombin-induced platelet aggregation of human blood. Furthermore, analysis of human umbilical vein endothelial cells, leukocytes, and platelets revealed that CNP selectively attenuates expression of P-selectin. Thus, CNP is a modulator of acute inflammation in the blood vessel wall characterized by leukocyte and platelet activation. These antiinflammatory effects appear to be mediated, at least in part, via suppression of P-selectin expression. These observations suggest that endothelial CNP might maintain an anti-atherogenic influence on the blood vessel wall and represent a target for therapeutic intervention in inflammatory cardiovascular disorders. endothelium | natriuretic peptide receptor type C | atherosclerosis | thrombosis

  18. Phospholipase C gamma mediates endogenous brain-derived neurotrophic factor-regulated calcitonin gene-related peptide expression in colitis-induced visceral pain

    PubMed Central

    Hashmi, Fiza; Liu, Miao; Shen, Shanwei

    2016-01-01

    Background Visceral hypersensitivity is a complex pathophysiological paradigm with unclear mechanisms. Primary afferent neuronal plasticity marked by alterations in neuroactive compounds such as calcitonin gene-related peptide is suggested to underlie the heightened sensory responses. Signal transduction that leads to calcitonin gene-related peptide expression thereby sensory neuroplasticity during colitis remains to be elucidated. Results In a rat model with colitis induced by 2,4,6-trinitrobenzene sulfonic acid, we found that endogenously elevated brain-derived neurotrophic factor elicited an up-regulation of calcitonin gene-related peptide in the lumbar L1 dorsal root ganglia. At seven days of colitis, neutralization of brain-derived neurotrophic factor with a specific brain-derived neurotrophic factor antibody reversed calcitonin gene-related peptide up-regulation in the dorsal root ganglia. Colitis-induced calcitonin gene-related peptide transcription was also inhibited by brain-derived neurotrophic factor antibody treatment. Signal transduction studies with dorsal root ganglia explants showed that brain-derived neurotrophic factor-induced calcitonin gene-related peptide expression was mediated by the phospholipase C gamma, but not the phosphatidylinositol 3-kinase/Akt or the mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway. Application of PLC inhibitor U73122 in vivo confirmed that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide up-regulation in the dorsal root ganglia was regulated by the phospholipase C gamma pathway. In contrast, suppression of the phosphatidylinositol 3-kinase activity in vivo had no effect on colitis-induced calcitonin gene-related peptide expression. During colitis, calcitonin gene-related peptide also co-expressed with phospholipase C gamma but not with p-Akt. Calcitonin gene-related peptide up-regulation during colitis correlated to the activation

  19. Overcoming the toxicity of membrane peptide expression in bacteria by upstream insertion of Asp-Pro sequence.

    PubMed

    Montigny, Cédric; Penin, François; Lethias, Claire; Falson, Pierre

    2004-01-28

    Transmembrane (TM) peptides often induce toxic effects when expressed in bacteria, probably due to membrane destabilization. We report here that in the case of the TM domains of hepatitis C virus (HCV) E1 and E2 envelope proteins, which are both particularly toxic for the bacteria, the insertion of the Asp-Pro (DP) sequence dramatically reduced their toxicities and promoted their expressions when produced as glutathione S-transferase (GST) GST-DP-TM chimeras. Subcellular fractionation showed that these chimeras co-sediment with the membrane fraction and contain active GST that could be solubilized with a mild detergent. Surprisingly, immuno-gold electron microscopy clearly showed that such chimeras are not localized in the membrane but in the cytosol. We thus postulate that they likely form proteo-lipidic aggregates, which prevent the bacteria from toxicity by sequestering the TM part of the chimeras. The reduction of toxicity in the presence of the Asp-Pro sequence is possibly due to Asp's negative charge that probably disadvantages the binding of the TM peptides to the membrane. In addition, the structural features of Pro residue could promote the formation of chimera aggregates. PMID:14757220

  20. Transgenic Brassica juncea plants expressing MsrA1, a synthetic cationic antimicrobial peptide, exhibit resistance to fungal phytopathogens.

    PubMed

    Rustagi, Anjana; Kumar, Deepak; Shekhar, Shashi; Yusuf, Mohd Aslam; Misra, Santosh; Sarin, Neera Bhalla

    2014-06-01

    Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44-62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56-71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants.

  1. Transgenic Brassica juncea plants expressing MsrA1, a synthetic cationic antimicrobial peptide, exhibit resistance to fungal phytopathogens.

    PubMed

    Rustagi, Anjana; Kumar, Deepak; Shekhar, Shashi; Yusuf, Mohd Aslam; Misra, Santosh; Sarin, Neera Bhalla

    2014-06-01

    Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44-62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56-71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants. PMID:24452332

  2. CP-10, a chemotactic peptide, is expressed in lesions of experimental autoimmune encephalomyelitis, neuritis, uveitis and in C6 gliomas.

    PubMed

    Deininger, M H; Zhao, Y; Schluesener, H J

    1999-01-01

    CP-10 (chemotactic protein of m.w. 10,000) is a member of the S100 superfamily of Ca2+ binding peptides, which has potent chemotactic activity for murine and human myeloid cells. Here we report on the generation of monoclonal antibodies against CP-10 and accumulation of CP-10+ cells during experimental autoimmune encephalomyelitis (EAE), neuritis (EAN), uveitis (EAU) and in experimentally transplanted C6 gliomas. During acute inflammation, CP-10 is mainly expressed by large ED1+ monocytic perivascular cells that accumulate at days 11-14. CP-10+ cells are predominantly located in areas of cellular infiltration but are as well found in the meninges and infiltrating the brain parenchyma. In transplanted gliomas, CP-10+ cells are located exclusively within the tumor parenchyma. Using double labeling experiments, other cells participating in the inflammatory reaction were found to express CP-10, like few lymphoblastic W3/13+ cells in the vicinity of the inflammatory infiltrate.

  3. Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli

    PubMed Central

    Saez, Natalie J.; Seshadri, Radha; Lau, Ho Yee; Bende, Niraj S.; Undheim, Eivind A. B.; Rash, Lachlan D.; Mobli, Mehdi; King, Glenn F.

    2013-01-01

    Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2–6 disulfide bonds. PMID:23667680

  4. Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli.

    PubMed

    Klint, Julie K; Senff, Sebastian; Saez, Natalie J; Seshadri, Radha; Lau, Ho Yee; Bende, Niraj S; Undheim, Eivind A B; Rash, Lachlan D; Mobli, Mehdi; King, Glenn F

    2013-01-01

    Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds.

  5. Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria

    PubMed Central

    Herrera Diaz, Areli; Kovacs, Izabella; Lindermayr, Christian

    2016-01-01

    Antimicrobial peptides (AMPs) are small peptides with less than 50 amino acids and are part of the innate immune response in almost all organisms, including bacteria, vertebrates, invertebrates and plants. AMPs are active against a broad-spectrum of pathogens. The inducible expression of AMPs in plants is a promising approach to combat plant pathogens with minimal negative side effects, such as phytotoxicity or infertility. In this study, inducible expression of the de-novo designed AMP SP1-1 in Micro Tom tomato protected tomato fruits against bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria. The peptide SP1-1 was targeted to the apoplast which is the primary infection site for plant pathogens, by fusing SP1-1 peptide to the signal peptide RsAFP1 of radish (Raphanus sativus). The pathogen inducibility of the expression was enabled by using an optimized inducible 4XW2/4XS promoter. As a result, the tomato fruits of independently generated SP1-1 transgenic lines were significantly more resistant to X. campestris pv. vesicatoria than WT tomato fruits. In transgenic lines, bacterial infection was reduced up to 65% in comparison to the infection of WT plants. Our study demonstrates that the combination of the 4XW2/4XS cis-element from parsley with the synthetic antimicrobial peptide SP1-1 is a good alternative to protect tomato fruits against infections with X. campestris pv. vesicatoria. PMID:27706237

  6. Heterologous expression and purification of plantaricin NC8, a two-peptide bacteriocin against Salmonella spp. from Lactobacillus plantarum ZJ316.

    PubMed

    Jiang, Han; Li, Ping; Gu, Qing

    2016-11-01

    Bacteriocin, which is produced by lactic acid bacteria (LAB), has the potential to act as natural preservatives in the food industry. To develop strategies to overproduce such peptides, plantaricin NC8, a class IIb LAB bacteriocin that consists of two peptides, PLNC8α and PLNC8β, was successfully heterologously expressed in Escherichia coli BL21 (DE3). PLNC8α and PLNC8β peptides were expressed as His6-tag fusion proteins and were separated by Ni(2+) chelating affinity chromatography. To get the PLNC8α and PLNC8β peptides without extra amino acids in the N-terminus, the fusion proteins were cleaved by enterokinase and further purified using the Ni-NTA Sefinose™ Resin Kit. The molecular masses of peptides were checked using Tricine-SDS-PAGE and MALDI-TOF-MS. The yield of purified PLNC8α was around 2-2.5 mg/L, and the yield of PLNC8β was around 1.5-2 mg/L. The antimicrobial spectrum of cleaved peptides was detected and the synergistic action of PLNC8α and PLNC8β was preliminarily confirmed. It was found that E. coli was a suitable host for heterologous expression of plantaricin NC8 with a significant yield. Importantly, the bacteriocin appeared to be very active for controlling and inhibiting the food-borne pathogenic Gram-negative bacteria Salmonella spp., and might be useful as a natural preservative candidate. PMID:27373940

  7. Oral Tolerance Induction in Experimental Autoimmune Encephalomyelitis with Candida utilis Expressing the Immunogenic MOG35-55 Peptide.

    PubMed

    Buerth, Christoph; Mausberg, Anne K; Heininger, Maximilian K; Hartung, Hans-Peter; Kieseier, Bernd C; Ernst, Joachim F

    2016-01-01

    Multiple sclerosis (MS) is an autoimmune disease that attacks myelinated axons in the central nervous system. Induction of oral tolerance is a potent mechanism to prevent autoimmunity. The food yeast Candida utilis was used to test the therapeutic potential of oral tolerance induction in an animal model of human multiple sclerosis (MS). We constructed a C. utilis strain, which displays a fusion peptide composed of the encephalitogenic MOG35-55 peptide and the C. utilis Gas1 cell wall protein on its surface.By immunizing mice with MOG35-55 peptide experimental autoimmune encephalomyelitis (EAE) was induced in a mouse model. Feeding of mice with C. utilis that expresses MOG35-55 peptide on its surface was started seven days prior to immunization and was continued for ten days. Control animals were treated with wild-type fungus or left untreated. Untreated mice developed first clinical symptoms ten days post immunization (p. i.) with an ascending paralysis reaching maximal clinical disability at day 18 to 20 p. i.. Treatment with the wild-type strain demonstrated comparable clinical symptoms. In contrast, oral gavage of MOG35-55-presenting fungus ameliorated the development of EAE. In addition, incidence as well as maximal clinical disease severity were significantly reduced. Interestingly, reduction of disease severity also occurred in animals treated with heat-inactivated C. utilis cells indicating that tolerance induction was independent of fungal viability. Better disease outcome correlated with reduced demyelination and cellular inflammation in the spinal cord, lower T cell proliferation against rechallenge with MOG35-55 and more regulatory T cells in the lymph nodes. Our data demonstrate successful that using the food approved fungus C. utilis presenting the immunogenic MOG35-55 peptide on its surface induced an oral tolerance against this epitope in EAE. Further studies will reveal the nature and extent of an anti-inflammatory environment established by the

  8. The glucagon-like peptide 2 receptor is expressed in enteric neurons and not in the epithelium of the intestine.

    PubMed

    Pedersen, Jens; Pedersen, Nis B; Brix, Sophie W; Grunddal, Kaare Villum; Rosenkilde, Mette M; Hartmann, Bolette; Ørskov, Cathrine; Poulsen, Steen S; Holst, Jens J

    2015-05-01

    Glucagon-like peptide 2 (GLP-2) is a potent intestinotrophic growth factor with therapeutic potential in the treatment of intestinal deficiencies. It has recently been approved for the treatment of short bowel syndrome. The effects of GLP-2 are mediated by specific binding of the hormone to the GLP-2 receptor (GLP-2R) which was cloned in 1999. However, consensus about the exact receptor localization in the intestine has never been established. By physical, chemical and enzymatic tissue fragmentation, we were able to divide rat jejunum into different compartments consisting of: (1) epithelium alone, (2) mucosa with lamina propria and epithelium, (3) the external muscle coat including myenteric plexus, (4) a compartment enriched for the myenteric plexus and (5) intestine without epithelium. Expression of Glp2r; chromogranin A; tubulin, beta 3; actin, gamma 2, smooth muscle, enteric and glial fibrillary acidic protein in these isolated tissue fractions was quantified with qRT-PCR. Expression of the Glp2r was confined to compartments containing enteric neurons and receptor expression was absent in the epithelium. Our findings provide evidence for the expression of the GLP-2R in intestinal compartments rich in enteric neurons and, importantly they exclude significant expression in the epithelium of rat jejunal mucosa.

  9. Expression pattern of the alpha-kafirin promoter coupled with a signal peptide from Sorghum bicolor L. Moench.

    PubMed

    Ahmad, Norazlina; Sant, Rajnesh; Bokan, Milovan; Steadman, Kathryn J; Godwin, Ian D

    2012-01-01

    Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

  10. Drosophila insulin-like peptide 1 (DILP1) is transiently expressed during non-feeding stages and reproductive dormancy

    PubMed Central

    Liu, Yiting; Liao, Sifang; Veenstra, Jan A.; Nässel, Dick R.

    2016-01-01

    The insulin/insulin-like growth factor signaling pathway is evolutionarily conserved in animals, and is part of nutrient-sensing mechanisms that control growth, metabolism, reproduction, stress responses, and lifespan. In Drosophila, eight insulin-like peptides (DILP1-8) are known, six of which have been investigated in some detail, whereas expression and functions of DILP1 and DILP4 remain enigmatic. Here we demonstrate that dilp1/DILP1 is transiently expressed in brain insulin producing cells (IPCs) from early pupa until a few days of adult life. However, in adult female flies where diapause is triggered by low temperature and short days, within a time window 0–10h post-eclosion, the dilp1/DILP1 expression remains high for at least 9 weeks. The dilp1 mRNA level is increased in dilp2, 3, 5 and dilp6 mutant flies, indicating feedback regulation. Furthermore, the DILP1 expression in IPCs is regulated by short neuropeptide F, juvenile hormone and presence of larval adipocytes. Male dilp1 mutant flies display increased lifespan and reduced starvation resistance, whereas in female dilp1 mutants oviposition is reduced. Thus, DILP1 is expressed in non-feeding stages and in diapausing flies, is under feedback regulation and appears to play sex-specific functional roles. PMID:27197757

  11. Expression pattern of the alpha-kafirin promoter coupled with a signal peptide from Sorghum bicolor L. Moench.

    PubMed

    Ahmad, Norazlina; Sant, Rajnesh; Bokan, Milovan; Steadman, Kathryn J; Godwin, Ian D

    2012-01-01

    Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP. PMID:22315514

  12. Drosophila insulin-like peptide 1 (DILP1) is transiently expressed during non-feeding stages and reproductive dormancy.

    PubMed

    Liu, Yiting; Liao, Sifang; Veenstra, Jan A; Nässel, Dick R

    2016-01-01

    The insulin/insulin-like growth factor signaling pathway is evolutionarily conserved in animals, and is part of nutrient-sensing mechanisms that control growth, metabolism, reproduction, stress responses, and lifespan. In Drosophila, eight insulin-like peptides (DILP1-8) are known, six of which have been investigated in some detail, whereas expression and functions of DILP1 and DILP4 remain enigmatic. Here we demonstrate that dilp1/DILP1 is transiently expressed in brain insulin producing cells (IPCs) from early pupa until a few days of adult life. However, in adult female flies where diapause is triggered by low temperature and short days, within a time window 0-10h post-eclosion, the dilp1/DILP1 expression remains high for at least 9 weeks. The dilp1 mRNA level is increased in dilp2, 3, 5 and dilp6 mutant flies, indicating feedback regulation. Furthermore, the DILP1 expression in IPCs is regulated by short neuropeptide F, juvenile hormone and presence of larval adipocytes. Male dilp1 mutant flies display increased lifespan and reduced starvation resistance, whereas in female dilp1 mutants oviposition is reduced. Thus, DILP1 is expressed in non-feeding stages and in diapausing flies, is under feedback regulation and appears to play sex-specific functional roles. PMID:27197757

  13. A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: cloning, heterologous expression and immunological cross-reactivity approaches.

    PubMed

    Matsubara, Fernando Hitomi; Gremski, Luiza Helena; Meissner, Gabriel Otto; Constantino Lopes, Eduardo Soares; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Silvio Sanches

    2013-09-01

    The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3-45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3-10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494 (DE3) cells. The peptide was expressed by IPTG induction for 4 h at 30 °C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.

  14. Expressions of Antimicrobial Peptides LL-37, Human Beta Defensin-2 and -3 in the Lesions of Cutaneous Tuberculosis and Tuberculids

    PubMed Central

    Zhao, Zheng; Mu, Zhang-Lei; Liu, Xi-Wan; Liu, Xiao-Jing; Jia, Jun; Cai, Lin; Zhang, Jian-Zhong

    2016-01-01

    Background: Antimicrobial peptides, including cathelicidin LL-37, human beta defensin (HBD)-2, and HBD-3, are important elements of the innate immune response and involved in modulation of the adaptive immunity, and they also play an important role in cutaneous defense against Mycobacterium tuberculosis. Methods: The fresh skin tissues and paraffin-embedded biopsy samples from three cutaneous tuberculosis, two tuberculids, and ten healthy individuals were collected. The expressions of LL-37, HBD-2, and HBD-3 mRNA in the lesions of three cutaneous tuberculosis and two tuberculids were detected by quantitative real-time polymerase chain reaction; the protein expressions were detected by immunohistochemistry and Western blotting methods. Results: The expressions of LL-37 mRNA and protein in the lesions of cutaneous tuberculosis and tuberculids were similar to that of normal skin. The expression of HBD-2 mRNA had an increasing trend in the lesions of cutaneous tuberculosis and tuberculids compared with that of normal skin; however, the expression of HBD-2 protein in the lesions of cutaneous tuberculosis had a decreasing trend compared with that of normal skin, and the expression of HBD-2 protein in the lesions of tuberculids was similar to that of normal skin. The expressions of HBD-3 mRNA and protein in lesions of cutaneous tuberculosis and tuberculids were similar to that of normal skin. Conclusions: Our study indicated that the expression of HBD-2 and HBD-3 mRNA and protein in lesions of cutaneous tuberculosis may be not consistent with that of tuberculids. However, an inherent limitation of the present study was that the sample size was small, and the roles and regulation mechanisms of LL-37, HBD-2, and HBD-3 in cutaneous tuberculosis and tuberculids need to be further investigated. PMID:26960373

  15. A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's β-amyloid peptides from E. coli for NMR-based structural analysis.

    PubMed

    Sharma, Sudhir C; Armand, Tara; Ball, K Aurelia; Chen, Anna; Pelton, Jeffrey G; Wemmer, David E; Head-Gordon, Teresa

    2015-12-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aβ42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aβ42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants.

  16. Cell surface-expressed moesin-like receptor regulates T cell interactions with tissue components and binds an adhesion-modulating IL-2 peptide generated by elastase.

    PubMed

    Ariel, A; Hershkoviz, R; Altbaum-Weiss, I; Ganor, S; Lider, O

    2001-03-01

    The adhesion of leukocytes to the extracellular matrix (ECM) depends on their responses to variations in the chemotactic signals in their milieu, as well as on the functioning of cytoskeletal and context-specific receptors. Ezrin, radixin, and moesin constitute a family of proteins that link the plasma membrane to the actin cytoskeleton. The surface expression of moesin on T cells and its role in cell adhesion has not been fully elucidated. Recently, we found that IL-2 peptides generated by elastase modified the adhesion of activated T cells to ECM ligands. Here, we further examined the adhesion regulatory effects of EFLNRWIT, one of the IL-2 peptides, as well as the existence and putative function of its receptor on T cells. We found that when presented to T cells in the absence of another activator, the EFLNRWIT peptide induced cell adhesion to vessel wall and ECM components. Binding of a radiolabeled peptide to T cells, precipitation with the immobilized peptide, and amino acid sequencing of the precipitated protein revealed that EFLNRWIT exerts its function via a cell surface-expressed moesin-like moiety, whose constitutive expression on T cells was increased after activation. This notion was further supported by our findings that: 1) anti-moesin mAb inhibited the binding of T cells to the immobilized EFLNRWIT peptide, 2) immobilized recombinant moesin bound the IL-2 peptide, and 3) soluble moesin inhibited the EFLNRWIT-induced T cell adhesion to fibronectin. Interestingly, moesin appears to be generally involved in T cell responses to adhesion-regulating signals. Thus, the IL-2 peptide EFLNRWIT appears to exert its modulating capacities via an adhesion-regulating moesin-like receptor. PMID:11207255

  17. Recombinant expression of a GH12 β-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi.

    PubMed

    Picart, Pere; Orejas, Margarita; Pastor, F I Javier

    2016-08-01

    The β-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 β-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete. PMID:27339304

  18. Recombinant expression and solution structure of antimicrobial peptide aurelin from jellyfish Aurelia aurita

    SciTech Connect

    Shenkarev, Zakhar O.; Panteleev, Pavel V.; Balandin, Sergey V.; Finkina, Ekaterina I.; Arseniev, Alexander S.; Ovchinnikova, Tatiana V.

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Aurelin was overexpressed in Escherichia coli, and its spatial structure was studied by NMR. Black-Right-Pointing-Pointer Aurelin compact structure encloses helical regions cross-linked by three disulfide bonds. Black-Right-Pointing-Pointer Aurelin shows structural homology to the BgK and ShK toxins of sea anemones. Black-Right-Pointing-Pointer Aurelin binds to the anionic lipid vesicles, but does not interact with zwitterionic ones. Black-Right-Pointing-Pointer Aurelin binds to DPC micelle surface with moderate affinity via two helical regions. -- Abstract: Aurelin is a 40-residue cationic antimicrobial peptide isolated from the mezoglea of a scyphoid jellyfish Aurelia aurita. Aurelin and its {sup 15}N-labeled analogue were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant peptide was examined, and its spatial structure was studied by NMR spectroscopy. Aurelin represents a compact globule, enclosing one 3{sub 10}-helix and two {alpha}-helical regions cross-linked by three disulfide bonds. The peptide binds to anionic lipid (POPC/DOPG, 3:1) vesicles even at physiological salt concentration, it does not interact with zwitterionic (POPC) vesicles and interacts with the DPC micelle surface with moderate affinity via two {alpha}-helical regions. Although aurelin shows structural homology to the BgK and ShK toxins of sea anemones, its surface does not possess the 'functional dyad' required for the high-affinity interaction with the K{sup +}-channels. The obtained data permit to correlate the modest antibacterial properties and membrane activity of aurelin.

  19. Alpha7 nicotinic acetylcholine receptor expression by vascular smooth muscle cells facilitates the deposition of Abeta peptides and promotes cerebrovascular amyloid angiopathy.

    PubMed

    Clifford, Peter M; Siu, Gilbert; Kosciuk, Mary; Levin, Eli C; Venkataraman, Venkateswar; D'Andrea, Michael R; Nagele, Robert G

    2008-10-01

    Deposition of beta-amyloid (Abeta) peptides in the walls of brain blood vessels, cerebral amyloid angiopathy (CAA), is common in patients with Alzheimer's disease (AD). Previous studies have demonstrated Abeta peptide deposition among vascular smooth muscle cells (VSMCs), but the source of the Abeta and basis for its selective deposition in VSMCs are unknown. In the present study, we examined the deposition patterns of Abeta peptides, Abeta40 and Abeta42, within the cerebrovasculature of AD and control patients using single- and double-label immunohistochemistry. Abeta40 and Abeta42 were abundant in VSMCs, especially in leptomeningeal arteries and their initial cortical branches; in later-stage AD brains this pattern extended into the microvasculature. Abeta peptide deposition was linked to loss of VSMC viability. Perivascular leak clouds of Abeta-positive material were associated primarily with arterioles. By contrast, control brains possessed far fewer Abeta42- and Abeta40-immunopositive blood vessels, with perivascular leak clouds of Abeta-immunopositive material rarely observed. We also demonstrate that VSMCs in brain blood vessels express the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), which has high binding affinity for Abeta peptides, especially Abeta42. These results suggest that the blood and blood-brain barrier permeability provide a major source of the Abeta peptides that gradually deposit in brain VSMCs, and the presence and abundance of the alpha7nAChR on VSMCs may facilitate the selective accumulation of Abeta peptides in these cells.

  20. Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus.

    PubMed

    Gottschalk, Sanne; Gottlieb, Caroline T; Vestergaard, Martin; Hansen, Paul R; Gram, Lone; Ingmer, Hanne; Thomsen, Line E

    2015-12-01

    The rapid rise in antibiotic-resistant pathogens is causing increased health concerns, and consequently there is an urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs), which have been isolated from a wide range of organisms, represent a very promising class of novel antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl(2) concentrations and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus-encoded natural tolerance mechanisms included peptide cleavage and the addition of positive charge to the cell surface, both of which minimized the antimicrobial activity of FL9. Our results add new information about FL9 and its effect on S. aureus, which may aid in the future development of analogues with improved therapeutic potential.

  1. Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections

    PubMed Central

    Lee, Seung-Bum; Li, Baichuan; Jin, Shuangxia; Daniell, Henry

    2012-01-01

    Summary Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC101 and PG1 accumulated up to 32%–38% and 17%~26% of the total soluble protein. Both RC101 and PG1 were cleaved from GFP by corresponding proteases in vitro, and Factor Xa–like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6-fold higher yield of RC101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to tobacco mosaic virus infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further preclinical studies. PMID:20553419

  2. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori.

    PubMed

    Wang, Yuancheng; Wang, Feng; Wang, Riyuan; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas.

  3. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori

    PubMed Central

    Wang, Yuancheng; Wang, Feng; Wang, Riyuan; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas. PMID:26537835

  4. Parathyroid hormone-related peptide (PTHrP): prokaryotic expression, purification, and preparation of a polyclonal antibody.

    PubMed

    Zheng, H L; Li, H; Sun, Y S; Yang, Z Y; Yu, Q

    2014-01-01

    Parathyroid hormone-related peptide (PTHrP) plays important roles in promoting cancer occurrence and in the development of bone metastases. To increase our knowledge of the biological functions of PTHrP, the prokaryotic expression vector pET-PTHrP was successfully constructed and the His-PTHrP fusion protein was expressed in Escherichia coli. Anti-PTHrP polyclonal antibody was then prepared from rabbits. Finally, the goat tissue expression profile of PTHrP was analyzed by Western blot with the anti-PTHrP polyclonal antibody. The results showed that the expression of PTHrP in goat mammary glands was significantly higher than that in other organs. This indicates that PTHrP may play important roles in the goat mammary gland. The antibody prepared will be a useful tool for detecting PTHrP and will be valuable in future studies investigating the role of PTHrP in calcium metabolism in the goat model. PMID:25158263

  5. Altered vasoactive intestinal peptides expression in irritable bowel syndrome patients and rats with trinitrobenzene sulfonic acid-induced colitis

    PubMed Central

    Del Valle-Pinero, Arseima Y; Sherwin, LeeAnne B; Anderson, Ethan M; Caudle, Robert M; Henderson, Wendy A

    2015-01-01

    AIM: To investigate the vasoactive intestinal peptides (VIP) expression in irritable bowel syndrome (IBS) and trinitrobenzene sulfonic acid (TNBS) induced colitis. METHODS: The VIP gene expression and protein plasma levels were measured in adult participants (45.8% male) who met Rome III criteria for IBS for longer than 6 mo and in a rat model of colitis as induced by TNBS. Plasma and colons were collected from naïve and inflamed rats. Markers assessing inflammation (i.e., weight changes and myeloperoxidase levels) were assessed on days 2, 7, 14 and 28 and compared to controls. Visceral hypersensitivity of the rats was assessed with colo-rectal distension and mechanical threshold testing on hind paws. IBS patients (n = 12) were age, gender, race, and BMI-matched with healthy controls (n = 12). Peripheral whole blood and plasma from fasting participants was collected and VIP plasma levels were assayed using a VIP peptide-enzyme immunoassay. Human gene expression of VIP was analyzed using a custom PCR array. RESULTS: TNBS induced colitis in the rats was confirmed with weight loss (13.7 ± 3.2 g) and increased myeloperoxidase activity. Visceral hypersensitivity to colo-rectal distension was increased in TNBS treated rats up to 21 d and resolved by day 28. Somatic hypersensitivity was also increased up to 14 d post TNBS induction of colitis. The expression of an inflammatory marker myeloperoxidase was significantly elevated in the intracellular granules of neutrophils in rat models following TNBS treatment compared to naïve rats. This confirmed the induction of inflammation in rats following TNBS treatment. VIP plasma concentration was significantly increased in rats following TNBS treatment as compared to naïve animals (P < 0.05). Likewise, the VIP gene expression from peripheral whole blood was significantly upregulated by 2.91-fold in IBS patients when compared to controls (P < 0.00001; 95%CI). VIP plasma protein was not significantly different when compared with

  6. The cytoplasmic peptide:N-glycanase (NGLY1) - Structure, expression and cellular functions.

    PubMed

    Suzuki, Tadashi; Huang, Chengcheng; Fujihira, Haruhiko

    2016-02-10

    NGLY1/Ngly1 is a cytosolic peptide:N-glycanase, i.e. de-N-glycosylating enzyme acting on N-glycoproteins in mammals, generating free, unconjugated N-glycans and deglycosylated peptides in which the N-glycosylated asparagine residues are converted to aspartates. This enzyme is known to be involved in the quality control system for the newly synthesized glycoproteins in the endoplasmic reticulum (ER). In this system, misfolded (glyco)proteins are retrotranslocated to the cytosol, where the 26S proteasomes play a central role in degrading the proteins: a process referred to as ER-associated degradation or ERAD in short. PNGase-mediated deglycosylation is believed to facilitate the efficient degradation of some misfolded glycoproteins. Human patients harboring mutations of NGLY1 gene (NGLY1-deficiency) have recently been discovered, clearly indicating the functional importance of this enzyme. This review summarizes the current state of our knowledge on NGLY1 and its gene product in mammalian cells. PMID:26611529

  7. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

    PubMed

    Große-Kreul, Jan; Busch, Maike; Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma. PMID:27626280

  8. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

    PubMed

    Große-Kreul, Jan; Busch, Maike; Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.

  9. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines

    PubMed Central

    Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma. PMID:27626280

  10. Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

    PubMed Central

    Garrett, J E; Collard, M W; Douglass, J O

    1989-01-01

    The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed. Images PMID:2573832

  11. Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle.

    PubMed

    Berghuis, Lesley; Abdelaziz, Khaled Taha; Bierworth, Jodi; Wyer, Leanna; Jacob, Gabriella; Karrow, Niel A; Sharif, Shayan; Clark, Mary Ellen; Caswell, Jeff L

    2014-10-12

    Bovine respiratory disease is a complex of bacterial and viral infections of economic and welfare importance to the beef industry. Although tracheal antimicrobial peptide (TAP) has microbicidal activity against bacterial pathogens causing bovine respiratory disease, risk factors for bovine respiratory disease including BVDV and stress (glucocorticoids) have been shown to inhibit the induced expression of this gene. Lipopolysaccharide is known to stimulate TAP gene expression, but the maximum effect is only observed after 16 h of stimulation. The present study investigated other agonists of TAP gene expression in primary cultures of bovine tracheal epithelial cells. PCR analysis of unstimulated tracheal epithelial cells, tracheal tissue and lung tissue each showed mRNA expression for Toll-like receptors (TLRs) 1-10. Quantitative RT-PCR analysis showed that Pam3CSK4 (an agonist of TLR1/2) and interleukin (IL)-17A significantly induced TAP gene expression in tracheal epithelial cells after only 4-8 h of stimulation. Flagellin (a TLR5 agonist), lipopolysaccharide and interferon-α also had stimulatory effects, but little or no response was found with class B CpG ODN 2007 (TLR9 agonist) or lipoteichoic acid (TLR2 agonist). The use of combined agonists had little or no enhancing effect above that of single agonists. Thus, Pam3CSK4, IL-17A and lipopolysaccharide rapidly and significantly induce TAP gene expression, suggesting that these stimulatory pathways may be of value for enhancing innate immunity in feedlot cattle at times of susceptibility to disease.

  12. Somatostatin receptor expression in small cell lung cancer as a prognostic marker and a target for peptide receptor radionuclide therapy

    PubMed Central

    Lapa, Constantin; Hänscheid, Heribert; Wild, Vanessa; Pelzer, Theo; Schirbel, Andreas; Werner, Rudolf A.; Droll, Sabine; Herrmann, Ken; Buck, Andreas K.; Lückerath, Katharina

    2016-01-01

    Despite initial responsiveness to both chemotherapy and radiotherapy, small cell lung cancer (SCLC) commonly relapses within months. Although neuroendocrine characteristics may be difficult to demonstrate in individual cases, a relevant expression of somatostatin receptors (SSTR) on the cell surface has been described. We aimed to evaluate the prognostic value of SSTR-expression in advanced SCLC. We further examined pre-requisites for successful peptide receptor radionuclide therapy (PRRT). 21 patients with extensive stage SCLC were enrolled. All patients underwent positron emission tomography/computed tomography (PET/CT) with 68Ga-DOTATATE to select patients for SSTR-directed therapy. PET scans were visually and semi-quantitatively assessed and compared to SSTR2a and SSTR5 expression in biopsy samples. Peak standardized uptake values (SUVpeak) of tumors as well as tumor-to-liver ratios were correlated to progression-free (PFS) and overall survival (OS). In 4/21 patients all SCLC lesions were PET-positive. 6/21 subjects were rated “intermediate” with the majority of lesions positive, the remaining 11/21 patients were PET-negative. PET-positivity correlated well with histologic SSTR2a, but not with SSTR5 expression. Neither PET-positivity nor SUVpeak were predictors of PFS or OS. In 4 patients with intensive SSTR2a-receptor expression, PRRT was performed with one partial response and one stable disease, respectively. SSTR-expression as detected by 68Ga-DOTATATE-PET and/or histology is not predictive of PFS or OS in patients with advanced SCLC. However, in patients exhibiting sufficient tracer uptake, PRRT might be a treatment option given its low toxicity and the absence of effective alternatives. PMID:26936994

  13. [Expression optimization and characterization of Tenebrio molitor antimicrobiol peptides TmAMP1m in Escherichia coli].

    PubMed

    Alimu, Reyihanguli; Mao, Xinfang; Liu, Zhongyuan

    2013-06-01

    To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside (IPTG). We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 degrees C for 4 h, there was the highest expression level of fusion protein HIS-TmAMP1m in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m had better stability and remained higher antibacterial activities when incubated at 100 degrees C for 10 h, repeated freeze thawing at -20 degrees C, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMP1m has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research. PMID:24063242

  14. Expression, purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris.

    PubMed

    Meng, De-Mei; Dai, Hong-Xia; Gao, Xiao-Fang; Zhao, Jing-Fang; Guo, Ya-Jun; Ling, Xiao; Dong, Bin; Zhang, Zi-Qi; Fan, Zhen-Chuan

    2016-11-01

    Mytichitin-A is an antimicrobial peptide isolated from the serum of Mytilus coruscus and is reported to inhibit bacterial growth as tested on several Gram-positive bacteria. To produce large quantity of Mytichitin-A to further investigate its biological activity, nucleotide sequence encoding a recombinant 6 × His-Mytichitin-A (rMytichitin-A) peptide was synthesized and inserted into the inducible yeast expression vector pPICZαA. With the availability of such an expression vector called pPICZαA-Mytichitin-A, we transformed Pichia pastoris GS115 cells with a SacI-linearized pPICZαA-Mytichitin-A by electroporation. Transgenic strains secreting rMytichitin-A with a molecular weight of approximate 10 KDa as expected were obtained. The optimal culture condition for rMytichitin-A expression was determined to be 1.0% methanol induction, 96 h incubation at 28 °C and the amount of rMytichitin-A reached 45.5 μg/ml. The percentage of rMytichitin-A was estimated to be 73.6% of the total protein. After rMytichitin-A was purified using nickel ions affinity chromatography, approximate 9.1 mg pure rMytichitin-A was obtained from 500 ml of cell culture medium with 97.8% purity. More importantly, both the culture supernatant and purified rMytichitin-A inhibited the growth of Gram-positive bacteria, especially Staphylococcus aureus and Bacillus subtilis with a minimum inhibition concentration of as low as 31 and 48 μg/ml, respectively. Differently from the native protein, however, the rMytichitin-A is not active against Gram-negative bacteria. Taken together, this is the first report on the heterologous expression of Mytichitin-A in P. pastoris. Our study showed that P. pastoris is an effective expression system for producing large quantities of biologically active Mytichitin-A for both research and application purposes. PMID:27389469

  15. Expression of Calcitonin Gene-related Peptide in Rat Pulp and Periodontal Tissues by Indirect Immunofluorescence Method

    PubMed Central

    Tao, Rui; Zhang, Mengjie; Cao, Xue; Wang, Hong; Xue, Lufeng; Wu, Meng

    2013-01-01

    The aim of this study was to investigate the expression of nerve fibers immunoreactive to calcitonin gene-related peptide (CGRP) in pulp and periodontal tissues of rats. Male Sprague-Dawley rats, aged 6 weeks, were sacrificed, and the jaws were excised, demineralized, and processed for indirect immunofluorescence staining. A considerably higher density of nerve fibers immunoreactive to CGRP was found in the dental pulp and gingiva than in periodontal ligament. The majority of pulpal CGRP immunopositive fibers that were located followed blood vessels parallel to the long axis of the root. A subodontoblastic network of fibers IR to CGRP was found in the coronal pulp in rat molars. In the periodontium, CGRP immunopositive fibers were mainly located in the periapical area and close to the alveolar bone. Gingiva was also well supplied with CGRP-IR nerves. PMID:24328744

  16. Nitric oxide induces gene expression of Jumonji and retinoblastoma 2 protein while reducing expression of atrial natriuretic peptide precursor type B in cardiomyocytes.

    PubMed

    Klassen, S S; Rabkin, S W

    2008-01-01

    Jumonji (JMJ, Jarid2), a prototypical member of the jumonji domain-containing protein family, plays a major role in embryonic cardiac development, but its role in the developed heart is unclear. Cardiomyocytes from neonatal mouse heart were treated in culture with NO donor SIN-1, 500 microM, for 2, 4, and 20 h. SIN-1 treatment was associated with a significant and 6.9 +/- 2.5 fold increase in jmj gene expression over all time points. The expression of jmj increased markedly and significantly 4.2 +/- 1.1 fold, 16.6 +/- 4.1 fold, and 2.7 +/- 0.3 fold, respectively, at time points 2 h, 4 h, and 20 h after treatment. The ability of the increase in gene expression to translate into an increase in cellular protein expression was ascertained by Western blotting, which showed an increase in the JMJ protein in whole-cell lysates. Because of the relationship of JMJ to Rb and ANP in the heart, gene expression of these proteins was also examined. SIN-1 produced a small but significant increase in Rb2, but not Rb1 or Rb-binding proteins 4, 6, or 7. In contrast, SIN-1 produced a marked and significant reduction in natriuretic peptide precursor type B but not type C to 0.24 +/- 0.09 fold of the control. These data suggest that JMJ may be a critical, previously unrecognized factor that mediates some of the cellular effects of NO, that NO may be able to increase JMJ in diseases associated with reduced JMJ expression.

  17. A shrimp C-type lectin inhibits proliferation of the hemolymph microbiota by maintaining the expression of antimicrobial peptides.

    PubMed

    Wang, Xian-Wei; Xu, Ji-Dong; Zhao, Xiao-Fan; Vasta, Gerardo Raul; Wang, Jin-Xing

    2014-04-25

    Some aquatic invertebrates such as shrimp contain low albeit stable numbers of bacteria in the circulating hemolymph. The proliferation of this hemolymph microbiota in such a nutrient-rich environment is tightly controlled in healthy animals, but the mechanisms responsible had remained elusive. In the present study, we report a C-type lectin (MjHeCL) from the kuruma shrimp (Marsupenaeus japonicus) that participates in restraining the hemolymph microbiota. Although the expression of MjHeCL did not seem to be modulated by bacterial challenge, the down-regulation of its expression by RNA interference led to proliferation of the hemolymph microbiota, ultimately resulting in shrimp death. This phenotype was rescued by the injection of recombinant MjHeCL, which restored the healthy status of the knockdown shrimp. A mechanistic analysis revealed that MjHeCL inhibited bacterial proliferation by modulating the expression of antimicrobial peptides. The key function of MjHeCL in the shrimp immune homeostasis might be related to its broader recognition spectrum of the hemolymph microbiota components than other lectins. Our study demonstrates the role of MjHeCL in maintaining the healthy status of shrimp and provides new insight into the biological significance of C-type lectins, a diversified and abundant lectin family in invertebrate species.

  18. Eukaryotic expression and purification of anti-epilepsy peptide of Buthus martensii Karsch and its protein interactions.

    PubMed

    Wang, Zongren; Wang, Wen; Shao, Zhongjun; Gao, Bifeng; Li, Junchang; Ma, Jing; Li, Jinghua; Che, Honglei; Zhang, Wei

    2009-10-01

    The Asian scorpion Buthus martensil Karsch is important in the Chinese traditional medicine where it is used for the treatment of some nervous system diseases. The anti-epilepsy peptide (AEP) is a 61-amino-acid polypeptide extracted from the venom of B. martensil Karsch. Research has confirmed that it has anti-epileptic effects on the rat model of epilepsy. In this experiment, a cDNA library of AEP from the venom of B. martensil Karsch was constructed using RT-PCR; the primer was designed and used for the amplification. An expression vector of AEP was constructed using Pichia pastoris. Vector expression was induced, and protein purification was then performed. Bolting of the interaction molecule of AEP was by His pull down. Experimental results indicate high AEP expression, and the obtained protein was purified and compared with the control group. Four conspicuous protein bands were observed, and mass chromatographic analysis indicated that the four proteins were synaptosomal-associated protein of 25 kDa (SNAP-25), glial fibrillary acidic protein (GFAP), Glutamic acid decarboxylase (GAD) and N-methyl-D: -aspartate (NMDA). Further, the four protein bands were verified by mammalian two-hybrid experiments and co-immunoprecipitation. AEP was found to interact with SNAP2 and NMDA. This provides experimental evidence for the mechanism of AEP's anti-epileptic action and for the manufacture of a novel type anti-epileptic drug. PMID:19370317

  19. Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility.

    PubMed

    Natsuga, Ken; Cipolat, Sara; Watt, Fiona M

    2016-01-01

    Mice lacking three epidermal barrier proteins-envoplakin, periplakin, and involucrin (EPI-/- mice)-have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4(+) T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance.

  20. Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility

    PubMed Central

    Natsuga, Ken; Cipolat, Sara; Watt, Fiona M.

    2016-01-01

    Mice lacking three epidermal barrier proteins—envoplakin, periplakin, and involucrin (EPI-/- mice)—have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4+ T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance. PMID:26763429

  1. Control of IGFBP-2 Expression by Steroids and Peptide Hormones in Vertebrates

    PubMed Central

    Hoeflich, Andreas; Wirthgen, Elisa; David, Robert; Classen, Carl Friedrich; Spitschak, Marion; Brenmoehl, Julia

    2014-01-01

    IGFBP-2 (1) has been described as a brain tumor oncogene (2) and is widely expressed in cancers from different origins (3–8). IGFBP-2 alone cannot cause malignant transformation, yet progression of brain tumors to higher grade (9) and also has been provided as a protective element in earlier stages of multistage colon carcinogenesis (10). Therefore, it is crucial to understand the factors that determine expression patterns of IGFBP-2 under normal and malignant conditions. The present review provides a comprehensive update of known factors that have an impact on expression of IGFBP-2. PMID:24778626

  2. A peptide that ameliorates lupus up-regulates the diminished expression of early growth response factors 2 and 3.

    PubMed

    Sela, Uri; Dayan, Molly; Hershkoviz, Rami; Lider, Ofer; Mozes, Edna

    2008-02-01

    Expansion of autoreactive T cells and their resistance to anergy was demonstrated in systemic lupus erythematosus (SLE). A pair of transcription factors, early growth response 2 (Egr-2) and 3 (Egr-3), are negative regulators of T cell activation that were shown to be important in anergy. A peptide (designated hCDR1 for human CDR1) based on the CDR-1 of an anti-DNA Ab ameliorated SLE in both induced and spontaneous lupus models. Our objectives were to determine the expression levels of Egr-2 and Egr-3 in autoreactive T cells following immunization with the lupus-inducing anti-DNA Ab that bears a common Id designated 16/6Id and also in a full-blown SLE and to determine the effect of hCDR1 on these transcription factors. We demonstrated diminished expression levels of Egr-2 and Egr-3 mRNA both early after immunization with the 16/6Id and in SLE-afflicted (NZB x NZW)F1 (New Zealand Black and New Zealand White) mice. Furthermore, by down-regulating Akt phosphorylation and up-regulating TGFbeta secretion, treatment with hCDR1 significantly up-regulated Egr-2 and Egr-3 expression. This was associated with an increased expression of the E3 ligase Cbl-b. Inhibition of Akt in T cells of immunized mice decreased, whereas silencing of the Egr-2 and Egr-3 in T cells of hCDR1-treated mice increased IFN-gamma secretion. Thus, hCDR1 down-regulates Akt phosphorylation, which leads to up-regulated expression of T cell Egr-2 and Egr-3, resulting in the inhibition of IFN-gamma secretion that is required for the maintenance of SLE. PMID:18209054

  3. Induction of porcine host defense peptide gene expression by short-chain fatty acids and their analogs.

    PubMed

    Zeng, Xiangfang; Sunkara, Lakshmi T; Jiang, Weiyu; Bible, Megan; Carter, Scott; Ma, Xi; Qiao, Shiyan; Zhang, Guolong

    2013-01-01

    Dietary modulation of the synthesis of endogenous host defense peptides (HDPs) represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections. However, HDP regulation by dietary compounds such as butyrate is species-dependent. To examine whether butyrate could induce HDP expression in pigs, we evaluated the expressions of a panel of porcine HDPs in IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and primary monocytes in response to sodium butyrate treatment by real-time PCR. We revealed that butyrate is a potent inducer of multiple, but not all, HDP genes. Porcine β-defensin 2 (pBD2), pBD3, epididymis protein 2 splicing variant C (pEP2C), and protegrins were induced markedly in response to butyrate, whereas pBD1 expression remained largely unaltered in any cell type. Additionally, a comparison of the HDP-inducing efficacy among saturated free fatty acids of different aliphatic chain lengths revealed that fatty acids containing 3-8 carbons showed an obvious induction of HDP expression in IPEC-J2 cells, with butyrate being the most potent and long-chain fatty acids having only a marginal effect. We further investigated a panel of butyrate analogs for their efficacy in HDP induction, and found glyceryl tributyrate, benzyl butyrate, and 4-phenylbutyrate to be comparable with butyrate. Identification of butyrate and several analogs with a strong capacity to induce HDP gene expression in pigs provides attractive candidates for further evaluation of their potential as novel alternatives to antibiotics in augmenting innate immunity and disease resistance of pigs. PMID:24023657

  4. Apolipoprotein A-IV inhibits AgRP/NPY neurons and activates POMC neurons in the arcuate nucleus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apolipoprotein A-IV (apoA-IV) in the brain potently suppresses food intake. However the mechanisms underlying its anorexigenic effects remain to be identified. We first examined the effects of apoA-IV on cellular activities in hypothalamic neurons that co-express agouti-related peptide (AgRP) and ne...

  5. Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain.

    PubMed

    Hughes, Stephen R; Dowd, Patrick F; Hector, Ronald E; Panavas, Tadas; Sterner, David E; Qureshi, Nasib; Bischoff, Kenneth M; Bang, Sookie S; Mertens, Jeffrey A; Johnson, Eric T; Li, Xin-Liang; Jackson, John S; Caughey, Robert J; Riedmuller, Steven B; Bartolett, Scott; Liu, Siqing; Rich, Joseph O; Farrelly, Philip J; Butt, Tauseef R; Labaer, Joshua; Cotta, Michael A

    2008-09-01

    New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin alpha-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction.

  6. The IDA/IDA-LIKE and PIP/PIP-LIKE gene families in Arabidopsis: phylogenetic relationship, expression patterns, and transcriptional effect of the PIPL3 peptide

    PubMed Central

    Vie, Ane Kjersti; Najafi, Javad; Liu, Bin; Winge, Per; Butenko, Melinka A.; Hornslien, Karina S.; Kumpf, Robert; Aalen, Reidunn B.; Bones, Atle M.; Brembu, Tore

    2015-01-01

    Peptide ligands play crucial roles in the life cycle of plants by modulating the innate immunity against pathogens and regulating growth and developmental processes. One well-studied example is INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which controls floral organ abscission and lateral root emergence in Arabidopsis thaliana. IDA belongs to a family of five additional IDA-LIKE (IDL) members that have all been suggested to be involved in regulation of Arabidopsis development. Here we present three novel members of the IDL subfamily and show that two of them are strongly and rapidly induced by different biotic and abiotic stresses. Furthermore, we provide data that the recently identified PAMP-INDUCED SECRETED PEPTIDE (PIP) and PIP-LIKE (PIPL) peptides, which show similarity to the IDL and C-TERMINALLY ENCODED PEPTIDE (CEP) peptides, are not only involved in innate immune response in Arabidopsis but are also induced by abiotic stress. Expression patterns of the IDA/IDL and PIP/PIPL genes were analysed using in silico data, qRT-PCR and GUS promoter lines. Transcriptomic responses to PIPL3 peptide treatment suggested a role in regulation of biotic stress responses and cell wall modification. PMID:26062745

  7. Expression of antimicrobial peptides and toll-like receptors is increased in tinea and pityriasis versicolor.

    PubMed

    Brasch, J; Mörig, A; Neumann, B; Proksch, E

    2014-03-01

    In superficial tinea and pityriasis versicolor, the causative fungi are for the most part confined to the stratum corneum which is barely reached by leukocytes. Therefore, a role of non-cellular components in the epidermal antifungal defence was suggested. To investigate the presence of such factors in these infections, the expression of human beta defensins 2 and 3 (hBD-2, hBD-3), RNase 7, psoriasin, toll-like receptors 2, 4 and 9 (TLR2, TLR4 and TLR9) and dectin 2 was analysed by use of immunostainings in skin biopsies. We found that hBD2, hBD3, psoriasin, RNase7, TLR2 and TLR4 were significantly more often expressed in distinct layers of lesional epidermis as compared with uninfected epidermis. In both infections but not in normal skin, hBD2 and hBD3 were commonly expressed within the stratum corneum and in the stratum granulosum. Similarly, psoriasin was seen more often in the upper skin layers of both infections as compared with normal skin. No significant differences between normal and infected skin were found for the expression of TLR9 and dectin 2. Our findings clearly show the expression of specific antimicrobial proteins and defence-related ligands in superficial tinea as well as in pityriasis versicolor, suggesting that these factors contribute to fungal containment.

  8. LPXRFamide peptide stimulates growth hormone and prolactin gene expression during the spawning period in the grass puffer, a semi-lunar synchronized spawner.

    PubMed

    Shahjahan, Md; Doi, Hiroyuki; Ando, Hironori

    2016-02-01

    Gonadotropin-inhibitory hormone (GnIH) plays as a multifunctional neurohormone that controls reproduction in birds and mammals. LPXRFamide (LPXRFa) peptide, the fish ortholog of GnIH, has been shown to regulate the secretion of not only gonadotropin (GTH) but also growth hormone (GH) and prolactin (PRL), which are potentially important for gonadal function. To investigate the role of LPXRFa peptide on reproduction of the grass puffer, which spawns in semilunar cycles, we examined changes in the levels of gh and prl expression over the several months during the reproductive cycle, and the effects of goldfish LPXRFa peptide-1 (gfLPXRFa-1) on their expression were examined using primary pituitary cultures. The expression levels of both gh and prl showed significant changes during the reproductive cycle in both sexes with one peak in the spawning and pre-spawning periods for gh and prl, respectively. Particularly, gh showed substantial increase in expression in the spawning and post-spawning periods, indicative of its essentiality in the advanced stage of reproduction. gfLPXRFa-1 stimulated the expression of both gh and prl but there was a marked difference in response between them: gfLPXRFa-1 stimulated gh expression at a relatively low dose but little effect was observed on prl. Combined with the previous results of daily and circadian oscillations of lpxrfa expression, the present results suggest that LPXRFa peptide is important in the control of the cyclic reproduction by serving as a multifunctional hypophysiotropic factor that regulates the expression of gh and prl as well as GTH subunit genes.

  9. Differential effects of methamphetamine on expression of neuropeptide Y mRNA in hypothalamus and on serum leptin and ghrelin concentrations in ad libitum-fed and schedule-fed rats.

    PubMed

    Crowley, W R; Ramoz, G; Keefe, K A; Torto, R; Kalra, S P; Hanson, G R

    2005-01-01

    Relatively little is known concerning the interaction of psychostimulants with hypothalamic neuropeptide systems or metabolic hormones implicated in regulation of energy balance. The present studies tested whether methamphetamine alters the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP), two important orexigenic neuropeptides, or proopiomelanocortin (POMC), the precursor for the anorexigenic peptide alpha-melanocyte-stimulating hormone, or the secretion of leptin, insulin and ghrelin, concomitant with inhibition of food intake. Female rats were either fed ad libitum (AL) or placed on a scheduled feeding (SF) regimen, with access to food limited to 4 h/day. Administration of (+/-)-methamphetamine (7.5 mg/kg, i.p.) 2 h prior to food presentation significantly inhibited food intake in SF animals, but did not affect intake in AL animals. In a separate study, AL and SF animals were killed just prior to expected food presentation, and expression of NPY, AgRP and POMC mRNAs in hypothalamus was determined using in situ hybridisation; concentrations of leptin, insulin and ghrelin in serum were determined with radioimmunoassays. In saline-treated, SF controls, NPY and AgRP mRNA expression in arcuate nucleus and serum ghrelin were significantly elevated, and serum leptin and insulin were significantly reduced. Methamphetamine reversed the up-regulation of NPY mRNA expression observed in the SF condition, without affecting AgRP mRNA or the serum concentrations of metabolic hormones. However, in AL animals, NPY mRNA expression in arcuate and dorsomedial nuclei was significantly increased by methamphetamine, which also reduced serum leptin and insulin and increased serum ghrelin concentrations. These findings suggest that the inhibition of NPY expression in SF animals may be a mechanism underlying the anorexigenic effect of methamphetamine seen in this condition. The increase in NPY expression produced by methamphetamine in AL animals may be mediated by the

  10. Gastrin Releasing Peptide Modulates Fast Delayed Rectifier Potassium Current in Per1-Expressing SCN Neurons

    PubMed Central

    Gamble, Karen L.; Kudo, Takashi; Colwell, Christopher S.; McMahon, Douglas G.

    2011-01-01

    The mammalian circadian clock in the suprachiasmatic nucleus (SCN) drives and maintains 24-h physiological rhythms, the phases of which are set by the local environmental light-dark cycle. Gastrin releasing peptide (GRP) communicates photic phase setting signals in the SCN by increasing neurophysiological activity of SCN neurons. Here, the ionic basis for persistent GRP-induced changes in neuronal activity was investigated in SCN slice cultures from Per1::GFP reporter mice during the early night. Recordings from Per1-fluorescent neurons in SCN slices several hours after GRP treatment revealed a significantly greater action potential frequency, a significant increase in voltage-activated outward current at depolarized potentials, and a significant increase in 4-aminopyridine (4-AP) sensitive fast delayed rectifier (fDR) potassium currents when compared to vehicle-treated slices. In addition, the persistent increase in spike rate following early night GRP application was blocked in SCN neurons from mice deficient in Kv3 channel proteins. Because fDR currents are regulated by the clock and are elevated in amplitude during the day, the present results support the model that GRP delays the phase of the clock during the early night by prolonging day-like membrane properties of SCN cells. Furthermore, these findings implicate fDR currents in the ionic basis for GRP-mediated entrainment of the primary mammalian circadian pacemaker. PMID:21454290

  11. Glugacon-like peptide-2: broad receptor expression, limited therapeutic effect on intestinal inflammation and novel role in liver regeneration.

    PubMed

    El-Jamal, Noura; Erdual, Edmone; Neunlist, Michel; Koriche, Dine; Dubuquoy, Caroline; Maggiotto, Francois; Chevalier, Julien; Berrebi, Dominique; Dubuquoy, Laurent; Boulanger, Eric; Cortot, Antoine; Desreumaux, Pierre

    2014-08-01

    The glucagon-like peptide 2 (GLP-2) is an intestinotrophic hormone with growth promoting and anti-inflammatory actions. However, the full biological functions of GLP-2 and the localization of its receptor (GLP-2R) remain controversial. Among cell lines tested, the expression of GLP-2R transcript was detected in human colonic myofibroblasts (CCD-18Co) and in primary culture of rat enteric nervous system but not in intestinal epithelial cell lines, lymphocytes, monocytes, or endothelial cells. Surprisingly, GLP-2R was expressed in murine (GLUTag), but not human (NCI-H716) enteroendocrine cells. The screening of GLP-2R mRNA in mice organs revealed an increasing gradient of GLP-2R toward the distal gut. An unexpected expression was detected in the mesenteric fat, mesenteric lymph nodes, bladder, spleen, and liver, particularly in hepatocytes. In two mice models of trinitrobenzene sulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced colitis, the colonic expression of GLP-2R mRNA was decreased by 60% compared with control mice. Also, GLP-2R mRNA was significantly downregulated in intestinal tissues of inflammatory bowel disease patients. Therapeutically, GLP-2 showed a weak restorative effect on intestinal inflammation during TNBS-induced colitis as assessed by macroscopic score and inflammatory markers. Finally, GLP-2 treatment accelerated mouse liver regeneration following partial hepatectomy as assessed by histological and molecular analyses. In conclusion, the limited therapeutic effect of GLP-2 on colonic inflammation dampens its utility in the management of severe inflammatory intestinal disorders. However, the role of GLP-2 in liver regeneration is a novelty that might introduce GLP-2 into the management of liver diseases and emphasizes on the importance of elucidating other extraintestinal functions of GLP-2. PMID:24875097

  12. A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.

    PubMed

    Orbán, Erika; Manea, Marilena; Marquadt, Andreas; Bánóczi, Zoltán; Csík, Gabriella; Fellinger, Erzsébet; Bosze, Szilvia; Hudecz, Ferenc

    2011-10-19

    Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

  13. Expression of the mouse MHC class Ib H2-T11 gene product, a paralog of H2-T23 (Qa-1) with shared peptide-binding specificity.

    PubMed

    Chen, Lili; Reyes-Vargas, Eduardo; Dai, Hu; Escobar, Hernando; Rudd, Brant; Fairbanks, Jared; Ho, Alexander; Cusick, Mathew F; Kumánovics, Attila; Delgado, Julio; He, Xiao; Jensen, Peter E

    2014-08-01

    The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with the highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted α3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP(+) and TAP(-) cells. Soluble T11D3 was generated by folding in vitro with Qa-1 determinant modifier, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qa-1 determinant modifier peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1-restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus, T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function. PMID:24958902

  14. Reduced expression of dermcidin, a peptide active against propionibacterium acnes, in sweat of patients with acne vulgaris.

    PubMed

    Nakano, Toshiaki; Yoshino, Takashi; Fujimura, Takao; Arai, Satoru; Mukuno, Akira; Sato, Naoya; Katsuoka, Kensei

    2015-09-01

    Dermcidin (DCD), an antimicrobial peptide with a broad spectrum of activity against bacteria such as Propionibacterum acnes, is expressed constitutively in sweat in the absence of stimulation due to injury or inflammation. The aim of this study was to determine the relationship between DCD expression and acne vulgaris associated with P. acnes. The antimicrobial activity of recombinant full-length DCD (50 μg/ml) was 97% against Escherichia coli and 100% against Staphylococcus aureus. Antimicrobial activity against P. acnes ranged from 68% at 50 μg/ml DCD to 83% at 270 μg/ml DCD. DCD concentration in sweat from patients with acne vulgaris (median 9.8 μg/ml, range 6.9-95.3 μg/ml) was significantly lower than in healthy subjects (median 136.7 μg/ml, range 45.4-201.6 μg/ml) (p = 0.001). DCD demonstrated concentration-dependent, but partial, microbicidal activity against P. acnes. These results suggest that reduced DCD concentration in sweat in patients with inflammatory acne may permit proliferation of P. acnes in pilosebaceous units, resulting in progression of inflammatory acne.

  15. Function and expression of sulfonylurea, adrenergic, and glucagon-like peptide 1 receptors in isolated porcine islets.

    PubMed

    Kelly, Amy C; Steyn, Leah V; Kitzmann, Jenna P; Anderson, Miranda J; Mueller, Kate R; Hart, Nathaniel J; Lynch, Ronald M; Papas, Klearchos K; Limesand, Sean W

    2014-01-01

    The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P < 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion.

  16. Soluble Beta-Amyloid Peptides, but Not Insoluble Fibrils, Have Specific Effect on Neuronal MicroRNA Expression

    PubMed Central

    Li, Jing Jing; Dolios, Georgia; Wang, Rong; Liao, Francesca-Fang

    2014-01-01

    Recent studies indicate that soluble β-amyloid (sAβ) oligomers, rather than their fibrillar aggregates, contribute to the pathogenesis of Alzheimer's disease (AD), though the mechanisms of their neurotoxicity are still elusive. Here, we demonstrate that sAβ derived from 7PA2 cells exert a much stronger effect on the regulation of a set of functionally validated microRNAs (miRNAs) in primary cultured neurons than the synthetic insoluble Aβ fibrils (fAβ). Synthetic sAβ peptides at a higher concentration present comparable effect on these miRNAs in our neuronal model. Further, the sAβ-induced miR-134, miR-145 and miR-210 expressions are fully reversed by two selective N-methyl-d-aspartate (NMDA) receptor inhibitors, but are neither reversed by insulin nor by forskolin, suggesting an NMDA receptor-dependent, rather than PI3K/AKT or PKA/CREB signaling dependent regulatory mechanism. In addition, the repression of miR-107 expression by the sAβ containing 7PA2 CM is likely involved multiple mechanisms and multiple players including NMDA receptor, N-terminally truncated Aβ and reactive oxygen species (ROS). PMID:24595404

  17. Peptide-Conjugated Gold Nanoprobe: Intrinsic Nanozyme-Linked Immunsorbant Assay of Integrin Expression Level on Cell Membrane.

    PubMed

    Gao, Liang; Liu, Meiqing; Ma, Guifu; Wang, Yaling; Zhao, Lina; Yuan, Qing; Gao, Fuping; Liu, Ru; Zhai, Jiao; Chai, Zhifang; Zhao, Yuliang; Gao, Xueyun

    2015-11-24

    Precisely quantifying the membrane protein expression level on cell surfaces is of vital importance for early cancer diagnosis and efficient treatment. We demonstrate that gold nanoparticle bioconjugated by a rationally designed peptide as nanoprobe possesses selective labeling and accurate quantification capacity of integrin GPIIb/IIIa on the human erythroleukemia cell line. Through selective recognition and marking of integrin, two-photon photoluminescence of the nanoprobe is exploited for direct observation of protein spatial distribution on cell membrane. More importantly, utilizing intrinsic enzyme-like catalysis property of the nanoprobe, the expression level of integrin on human erythroleukemia cells can be quantitatively counted in an amplified and reliable colorimetric assay without cell lysis and protein extraction process. In addition, the analysis of the correlation between the gold nanoparticle and the membrane protein via relevant inductively coupled plasma mass spectrometry measurement verifies the reliability of the new analytical method. It is anticipated that this facile and efficient strategy holds a great promise for a rapid, precise, and reliable quantification of interested functional membrane proteins on the cell surface.

  18. Blockage of the Neonatal Leptin Surge Affects the Gene Expression of Growth Factors, Glial Proteins, and Neuropeptides Involved in the Control of Metabolism and Reproduction in Peripubertal Male and Female Rats.

    PubMed

    Mela, Virginia; Díaz, Francisca; Lopez-Rodriguez, Ana Belen; Vázquez, María Jesús; Gertler, Arieh; Argente, Jesús; Tena-Sempere, Manuel; Viveros, María-Paz; Chowen, Julie A

    2015-07-01

    Leptin (Lep) is important in the development of neuroendocrine circuits involved in metabolic control. Because both Lep and metabolism influence pubertal development, we hypothesized that early changes in Lep signaling could also modulate hypothalamic (HT) systems involved in reproduction. We previously demonstrated that a single injection of a Lep antagonist (Antag) on postnatal day (PND)9, coincident with the neonatal Lep peak, induced sexually dimorphic modifications in trophic factors and markers of cell turnover and neuronal maturation in the HT on PND13. Here, our aim was to investigate whether the alterations induced by Lep antagonism persist into puberty. Accordingly, male and female rats were treated with a pegylated super Lep Antag from PND5 to PND9 and killed just before the normal appearance of external signs of puberty (PND33 in females and PND43 in males). There was no effect on body weight, but in males food intake increased, subcutaneous adipose tissue decreased and HT neuropeptide Y and Agouti-related peptide mRNA levels were reduced, with no effect in females. In both sexes, the Antag increased HT mRNA levels of the kisspeptin receptor, G protein-coupled recepter 54 (Gpr54). Expression of the Lep receptor, trophic factors, and glial markers were differently affected in the HT of peripubertal males and females. Lep production in adipose tissue was decreased in Antag-treated rats of both sexes, with production of other cytokines being differentially regulated between sexes. In conclusion, in addition to the long-term effects on metabolism, changes in neonatal Lep levels modifies factors involved in reproduction that could possibly affect sexual maturation.

  19. Effect of residual feed intake on hypothalamic gene expression and meat quality in Angus-sired cattle grown during the hot season.

    PubMed

    Perkins, S D; Key, C N; Marvin, M N; Garrett, C F; Foradori, C D; Bratcher, C L; Kriese-Anderson, L A; Brandebourg, T D

    2014-04-01

    The relationship between heat stress, meat quality, and residual feed intake (RFI) is unknown in growing steers. To address this issue, high RFI (HRFI) and low RFI (LRFI) individuals were compared by assessing RFI in 48 Angus-sired steers during a 70-d feeding trial conducted during July through September to identify steers with calculated RFI at least 2 SD apart. The association of RFI with indices of meat quality and expression of genes within hypothalamic and adipose tissue was then determined in LRFI and HRFI steers. While on test, feed intake was recorded daily with BW and hip heights recorded every 14 d. Ultrasound measurements of rib eye area (REA) and backfat (BF) were recorded initially and before harvest. Carcass and growth data were analyzed using a mixed model with RFI level (LRFI and HRFI) as the independent variable. The least square means for RFI were -1.2 and 0.99 kg DMI/d, respectively, for the LRFI and HRFI cohorts (P < 0.0001). Dry matter intake was higher for the HRFI individuals versus the LRFI steers (P < 0.0001) while on-test gain was not different (P < 0.95). Marbling score was greater in LRFI than HRFI steers (P < 0.05). However, there were no differences in REA (P < 0.53), BF (P < 0.65), yield grade (P < 0.24), or objective Hunter color measures between LRFI and HRFI steers indicating there was no consistent relationship between RFI and indices of meat quality. Hypothalamic neuropeptide Y (NPY), agouti related protein (AGRP), relaxin-3 (RLN3), melanocortin 3 receptor, and relaxin/insulin-like family peptide receptor 1 (RXFP1) mRNA were expressed 280, 185, 202, 183, and 163% greater, respectively (P < 0.01), while proopiomelanocortin (POMC) mRNA was expressed 42% lower in LRFI than HRFI animals (P < 0.05). Hypothalamic GnRH mRNA expression was 67% lower while gonadotropin inhibiting hormone (GnIH) mRNA was 209% higher in LRFI than HRFI animals (P < 0.01). Pituitary expression of FSHβ and LHβ correlated to hypothalamic GnRH levels (P < 0

  20. Effect of residual feed intake on hypothalamic gene expression and meat quality in Angus-sired cattle grown during the hot season.

    PubMed

    Perkins, S D; Key, C N; Marvin, M N; Garrett, C F; Foradori, C D; Bratcher, C L; Kriese-Anderson, L A; Brandebourg, T D

    2014-04-01

    The relationship between heat stress, meat quality, and residual feed intake (RFI) is unknown in growing steers. To address this issue, high RFI (HRFI) and low RFI (LRFI) individuals were compared by assessing RFI in 48 Angus-sired steers during a 70-d feeding trial conducted during July through September to identify steers with calculated RFI at least 2 SD apart. The association of RFI with indices of meat quality and expression of genes within hypothalamic and adipose tissue was then determined in LRFI and HRFI steers. While on test, feed intake was recorded daily with BW and hip heights recorded every 14 d. Ultrasound measurements of rib eye area (REA) and backfat (BF) were recorded initially and before harvest. Carcass and growth data were analyzed using a mixed model with RFI level (LRFI and HRFI) as the independent variable. The least square means for RFI were -1.2 and 0.99 kg DMI/d, respectively, for the LRFI and HRFI cohorts (P < 0.0001). Dry matter intake was higher for the HRFI individuals versus the LRFI steers (P < 0.0001) while on-test gain was not different (P < 0.95). Marbling score was greater in LRFI than HRFI steers (P < 0.05). However, there were no differences in REA (P < 0.53), BF (P < 0.65), yield grade (P < 0.24), or objective Hunter color measures between LRFI and HRFI steers indicating there was no consistent relationship between RFI and indices of meat quality. Hypothalamic neuropeptide Y (NPY), agouti related protein (AGRP), relaxin-3 (RLN3), melanocortin 3 receptor, and relaxin/insulin-like family peptide receptor 1 (RXFP1) mRNA were expressed 280, 185, 202, 183, and 163% greater, respectively (P < 0.01), while proopiomelanocortin (POMC) mRNA was expressed 42% lower in LRFI than HRFI animals (P < 0.05). Hypothalamic GnRH mRNA expression was 67% lower while gonadotropin inhibiting hormone (GnIH) mRNA was 209% higher in LRFI than HRFI animals (P < 0.01). Pituitary expression of FSHβ and LHβ correlated to hypothalamic GnRH levels (P < 0

  1. Sex and estrogen receptor expression influence opioid peptide levels in the mouse hippocampal mossy fiber pathway

    PubMed Central

    Van Kempen, Tracey A.; Kahlid, Sana; Gonzalez, Andreina D.; Spencer-Segal, Joanna L.; Tsuda, Mumeko C.; Ogawa, Sonoko; McEwen, Bruce S.; Waters, Elizabeth M.; Milner, Teresa A.

    2013-01-01

    The opioid peptides, dynorphin (DYN) and enkephalin (L-ENK) are contained in the hippocampal mossy fiber pathway where they modulate synaptic plasticity. In rats, the levels of DYN and L-ENK immunoreactivity (-ir) are increased when estrogen levels are elevated (Torres-Reveron et al. 2008 and 2009). Here, we used quantitative immunocytochemistry to examine whether opioid levels are similarly regulated in wildtype (WT) mice over the estrous cycle, and how these compared to males. Moreover, using estrogen receptor (ER) alpha and beta knockout mice (AERKO and BERKO, respectively), the present study examined the role of ERs in rapid, membrane-initiated (6 hr), or slower, nucleus-initiated (48 hr) estradiol effects on mossy fiber opioid levels. Unlike rats, the levels of DYN and L-ENK-ir did not change over the estrous cycle. However, compared to males, females had higher levels of DYN-ir in CA3a and L-ENK-ir in CA3b. In WT and BERKO ovariectomized (OVX) mice, neither DYN- nor L-ENK-ir changed following 6 or 48 hrs estradiol benzoate (EB) administration. However, DYN-ir significantly increased 48 hours after EB in the dentate gyrus (DG) and CA3b of AERKO mice only. These findings suggest that cyclic hormone levels regulate neither DYN nor L-ENK levels in the mouse mossy fiber pathway as they do in the rat. This may be due to species-specific differences in the mossy fiber pathway. However, in the mouse, DYN levels are regulated by exogenous EB in the absence of ERα possibly via an ERβ-mediated pathway requiring new gene transcription. PMID:23933204

  2. Sex and estrogen receptor expression influence opioid peptide levels in the mouse hippocampal mossy fiber pathway.

    PubMed

    Van Kempen, Tracey A; Kahlid, Sana; Gonzalez, Andreina D; Spencer-Segal, Joanna L; Tsuda, Mumeko C; Ogawa, Sonoko; McEwen, Bruce S; Waters, Elizabeth M; Milner, Teresa A

    2013-09-27

    The opioid peptides, dynorphin (DYN) and enkephalin (L-ENK) are contained in the hippocampal mossy fiber pathway where they modulate synaptic plasticity. In rats, the levels of DYN and L-ENK immunoreactivity (-ir) are increased when estrogen levels are elevated (Torres-Reveron et al., 2008, 2009). Here, we used quantitative immunocytochemistry to examine whether opioid levels are similarly regulated in wildtype (WT) mice over the estrous cycle, and how these compared to males. Moreover, using estrogen receptor (ER) alpha and beta knock-out mice (AERKO and BERKO, respectively), the present study examined the role of ERs in rapid, membrane-initiated (6 h), or slower, nucleus-initiated (48 h) estradiol effects on mossy fiber opioid levels. Unlike rats, the levels of DYN and L-ENK-ir did not change over the estrous cycle. However, compared to males, females had higher levels of DYN-ir in CA3a and L-ENK-ir in CA3b. In WT and BERKO ovariectomized (OVX) mice, neither DYN- nor L-ENK-ir changed following 6 or 48 h estradiol benzoate (EB) administration. However, DYN-ir significantly increased 48 h after EB in the dentate gyrus (DG) and CA3b of AERKO mice only. These findings suggest that cyclic hormone levels regulate neither DYN nor L-ENK levels in the mouse mossy fiber pathway as they do in the rat. This may be due to species-specific differences in the mossy fiber pathway. However, in the mouse, DYN levels are regulated by exogenous EB in the absence of ERα possibly via an ERβ-mediated pathway requiring new gene transcription.

  3. Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17.

    PubMed Central

    Allison, G E; Worobo, R W; Stiles, M E; Klaenhammer, T R

    1995-01-01

    The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 and is active against five other Lactobacillus species and Enterococcus faecalis. The genetic determinants encoding the lactacin F complex are organized in a 1-kb polycistronic operon which comprises three genes, lafA, lafX, and ORFZ (encoding the putative immunity protein). The lafA and lafX genes encode the bacteriocin precursors with N-terminal extensions characterized by a Gly-Gly-1*Xaa+1 cleavage site (*). The Gly-Gly motif is conserved in several other bacteriocins, including carnobacteriocins A, BM1, and B2. Carnobacterium piscicola LV17 produces carnobacteriocins which are active against Listeria monocytogenes and other lactic acid bacteria. In this study, the lactacin F operon was introduced into C. piscicola LV17. The transformants produced lactacin F concurrently with the carnobacteriocins. When the lafA and lafX genes were separated and cloned individually into LV17, production of either LafA or LafX by C. piscicola LV17 was detected by complementation with L. johnsonii clones producing LafX or LafA, respectively. Transformants of C. piscicola LV17 which produced lactacin F, LafA, or LafX, in combination with the carnobacteriocins, were assayed for an increased and expanded inhibitory spectrum. The recombinant organisms were only active against lactacin F- and carnobacteriocin-sensitive strains. A plasmidless derivative of LV17 which does not produce the carnobacteriocins failed to produce lactacin F, LafA, or LafX when transformed with the appropriate recombinant plasmids. The ability of C. piscicola LV17 to produce lactacin F demonstrates that the machinery for the carnobacteriocins is capable of processing and exporting bacteriocins from both systems. PMID:7747957

  4. Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide.

    PubMed

    Rasala, Beth A; Lee, Philip A; Shen, Zhouxin; Briggs, Steven P; Mendez, Michael; Mayfield, Stephen P

    2012-01-01

    Microalgae have recently received attention as a potential low-cost host for the production of recombinant proteins and novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expression of heterologous genes from the nuclear genome. Here we describe a nuclear expression strategy using the foot-and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistance in Chlamydomonas reinhardtii. We demonstrate that strains transformed with ble-2A-GFP are zeocin-resistant and accumulate high levels of GFP that is properly 'cleaved' at the FMDV 2A peptide resulting in monomeric, cytosolic GFP that is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expression vector to engineer the heterologous expression of the industrial enzyme, xylanase. We demonstrate that linking xyn1 expression to ble2A expression on the same open reading frame led to a dramatic (~100-fold) increase in xylanase activity in cells lysates compared to the unlinked construct. Finally, by inserting an endogenous secretion signal between the ble2A and xyn1 coding regions, we were able to target monomeric xylanase for secretion. The novel microalgae nuclear expression strategy described here enables the selection of transgenic lines that are efficiently expressing the heterologous gene-of-interest and should prove valuable for basic research as well as algal biotechnology. PMID:22937037

  5. The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis

    PubMed Central

    2014-01-01

    Background The nootropic neuroprotective peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has proved efficient in the therapy of brain stroke; however, the molecular mechanisms underlying its action remain obscure. Our genome-wide study was designed to investigate the response of the transcriptome of ischemized rat brain cortex tissues to the action of Semax in vivo. Results The gene-expression alteration caused by the action of the peptide Semax was compared with the gene expression of the “ischemia” group animals at 3 and 24 h after permanent middle cerebral artery occlusion (pMCAO). The peptide predominantly enhanced the expression of genes related to the immune system. Three hours after pMCAO, Semax influenced the expression of some genes that affect the activity of immune cells, and, 24 h after pMCAO, the action of Semax on the immune response increased considerably. The genes implicated in this response represented over 50% of the total number of genes that exhibited Semax-induced altered expression. Among the immune-response genes, the expression of which was modulated by Semax, genes that encode immunoglobulins and chemokines formed the most notable groups. In response to Semax administration, 24 genes related to the vascular system exhibited altered expression 3 h after pMCAO, whereas 12 genes were changed 24 h after pMCAO. These genes are associated with such processes as the development and migration of endothelial tissue, the migration of smooth muscle cells, hematopoiesis, and vasculogenesis. Conclusions Semax affects several biological processes involved in the function of various systems. The immune response is the process most markedly affected by the drug. Semax altered the expression of genes that modulate the amount and mobility of immune cells and enhanced the expression of genes that encode chemokines and immunoglobulins. In conditions of rat brain focal ischemia, Semax influenced the expression of genes that promote the formation and

  6. Temporal and Regional Expression of Glucose-Dependent Insulinotropic Peptide and Its Receptor in Spinal Cord Injured Rats.

    PubMed

    Marcos, Ana Beatriz W; Forner, Stefania; Martini, Alessandra C; Patrício, Eliziane S; Clarke, Julia R; Costa, Robson; Felix-Alves, João; Vieira, Vilberto José; de Andrade, Edinéia Lemos; Mazzuco, Tânia Longo; Calixto, João Batista; Figueiredo, Claudia Pinto

    2016-02-01

    Spinal cord injury (SCI) results in loss of movement, sensibility, and autonomic control at the level of the lesion and at lower parts of the body. Several experimental strategies have been used in attempts to increase endogenous mechanisms of neuroprotection, neuroplasticity, and repair, but with limited success. It is known that glucose-dependent insulinotropic peptide (GIP) and its receptor (GIPR) can enhance synaptic plasticity, neurogenesis, and axonal outgrowth. However, their role in the injury has never been studied. The aim of this study was to evaluate the changes in expression levels of both GIP and GIPR in acute and chronic phases of SCI in rats. Following SCI (2 to 24 h after damage), the rat spinal cord showed a lesion in which the epicenter had a cavity with hemorrhage and necrosis. Furthermore, the lesion cavity also showed ballooned cells 14 and 28 days after injury. We found that SCI induced increases in GIPR expression in areas neighboring the site of injury at 6 h and 28 days after the injury. Moreover, higher GIP expression was observed in these regions on day 28. Neuronal projections from the injury epicenter showed an increase in GIP immunoreactivity 24 h and 14 and 28 days after SCI. Interestingly, GIP was also found in progenitor cells at the spinal cord canal 24 h after injury, whereas both GIP and GIPR were present in progenitor cells at the injury epicenter 14 days after in SCI animals. These results suggest that GIP and its receptor might be implicated with neurogenesis and the repair process after SCI. PMID:26421658

  7. Expression of an Engineered Heterologous Antimicrobial Peptide in Potato Alters Plant Development and Mitigates Normal Abiotic and Biotic Responses

    PubMed Central

    Goyal, Ravinder K.; Hancock, Robert E. W.; Mattoo, Autar K.; Misra, Santosh

    2013-01-01

    Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield. PMID:24147012

  8. Expression of an engineered heterologous antimicrobial peptide in potato alters plant development and mitigates normal abiotic and biotic responses.

    PubMed

    Goyal, Ravinder K; Hancock, Robert E W; Mattoo, Autar K; Misra, Santosh

    2013-01-01

    Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield.

  9. Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: Evidence for the alternative splicing of a single-copy gene

    SciTech Connect

    Thiede, M.A.; Strewler, G.J.; Nissenson, R.A.; Rosenblatt, M.; Rodan, G.A. )

    1988-07-01

    A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study the authors obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3{prime} untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A){sup +} RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3{prime} untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene.

  10. Interleukin 13 Exposure Enhances Vitamin D-Mediated Expression of the Human Cathelicidin Antimicrobial Peptide 18/LL-37 in Bronchial Epithelial Cells

    PubMed Central

    van Sterkenburg, M. A. J. A.; Verhoosel, R. M.; Zuyderduyn, S.; Hiemstra, P. S.

    2012-01-01

    Vitamin D is an important regulator of the expression of antimicrobial peptides, and vitamin D deficiency is associated with respiratory infections. Regulating expression of antimicrobial peptides, such as the human cathelicidin antimicrobial peptide 18 (hCAP18)/LL-37, by vitamin D in bronchial epithelial cells requires local conversion of 25(OH)-vitamin D3 (25D3) into its bioactive metabolite, 1,25(OH)2-vitamin D3 (1,25D3), by CYP27B1. Low circulating vitamin D levels in childhood asthma are associated with more-severe exacerbations, which are often associated with infections. Atopic asthma is accompanied by Th2-driven inflammation mediated by cytokines such as interleukin 4 (IL-4) and IL-13, and the effect of these cytokines on vitamin D metabolism and hCAP18/LL-37 expression is unknown. Therefore, we investigated this with well-differentiated bronchial epithelial cells. To this end, cells were treated with IL-13 with and without 25D3, and expression of hCAP18/LL-37, CYP27B1, the 1,25D3-inactivating enzyme CYP24A1, and vitamin D receptor was assessed by quantitative PCR. We show that IL-13 enhances the ability of 25D3 to increase expression of hCAP18/LL-37 and CYP24A1. In addition, exposure to IL-13 resulted in increased CYP27B1 expression, whereas vitamin D receptor (VDR) expression was not significantly affected. The enhancing effect of IL-13 on 25D3-mediated expression of hCAP18/LL-37 was further confirmed using SDS-PAGE Western blotting and immunofluorescence staining. In conclusion, we demonstrate that IL-13 induces vitamin D-dependent hCAP18/LL-37 expression, most likely by increasing CYP27B1. These data suggest that Th2 cytokines regulate the vitamin D metabolic pathway in bronchial epithelial cells. PMID:23045480

  11. Expression of a Novel Antimicrobial Peptide Penaeidin4-1 in Creeping Bentgrass (Agrostis stolonifera L.) Enhances Plant Fungal Disease Resistance

    PubMed Central

    Zhou, Man; Hu, Qian; Li, Zhigang; Li, Dayong; Chen, Chin-Fu; Luo, Hong

    2011-01-01

    Background Turfgrass species are agriculturally and economically important perennial crops. Turfgrass species are highly susceptible to a wide range of fungal pathogens. Dollar spot and brown patch, two important diseases caused by fungal pathogens Sclerotinia homoecarpa and Rhizoctonia solani, respectively, are among the most severe turfgrass diseases. Currently, turf fungal disease control mainly relies on fungicide treatments, which raises many concerns for human health and the environment. Antimicrobial peptides found in various organisms play an important role in innate immune response. Methodology/Principal Findings The antimicrobial peptide - Penaeidin4-1 (Pen4-1) from the shrimp, Litopenaeus setiferus has been reported to possess in vitro antifungal and antibacterial activities against various economically important fungal and bacterial pathogens. In this study, we have studied the feasibility of using this novel peptide for engineering enhanced disease resistance into creeping bentgrass plants (Agrostis stolonifera L., cv. Penn A-4). Two DNA constructs were prepared containing either the coding sequence of a single peptide, Pen4-1 or the DNA sequence coding for the transit signal peptide of the secreted tobacco AP24 protein translationally fused to the Pen4-1 coding sequence. A maize ubiquitin promoter was used in both constructs to drive gene expression. Transgenic turfgrass plants containing different DNA constructs were generated by Agrobacterium-mediated transformation and analyzed for transgene insertion and expression. In replicated in vitro and in vivo experiments under controlled environments, transgenic plants exhibited significantly enhanced resistance to dollar spot and brown patch, the two major fungal diseases in turfgrass. The targeting of Pen4-1 to endoplasmic reticulum by the transit peptide of AP24 protein did not significantly impact disease resistance in transgenic plants. Conclusion/Significance Our results demonstrate the effectiveness

  12. Increased mRNA expression of selected antimicrobial peptides around ovulation and during inflammatory processes in the bovine endometrium postpartum.

    PubMed

    Ibrahim, M; Peter, S; Gärtner, M A; Michel, G; Jung, M; Einspanier, R; Gabler, C

    2016-11-01

    In the uterus, the first pathogen confrontations take place at the luminal endometrial epithelium. Therefore, it is required that these cells have the potential to recognize and respond to a bacterial infection. Antimicrobial peptides (AMP), part of the innate immune system in addition to cytokines, are principal effector molecules of mucosal immunity against pathogens. One important family of AMP that can permeabilize bacterial membranes is the beta-defensin (DEFB) family, which includes the following members: DEFB1, DEFB4A, and DEFB5, lingual AMP, and tracheal AMP. The bactericidal/permeability-increasing protein is also a cationic AMP that results in the death of bacteria. Another AMP family is the S100 calcium-binding protein (S100A) family including the following members: S100A8, S100A9, S100A11, and S100A12. These AMP exert their antimicrobial action through chelation of several ions. The aim of the present study was to evaluate mRNA expression patterns of selected AMP in bovine endometrial cells collected (1) at different stages of the estrous cycle (postovulatory, early-to-mid luteal, late luteal, and pre-ovulatory phase); (2) during the puerperium depending on uterine health status (healthy, subclinical, or clinical endometritis) starting on Day 24 to 30 postpartum for 3 weeks on a weekly basis; and (3) in vitro after co-culturing with Bacillus pumilus at three different multiplicities of infection (MOI 1, 5, and 10) up to 6 hours. The results reported that the mRNA expression of all candidate AMP, except DEFB1, S100A8, and S100A9, was estrous cycle dependent. In particular, around the time of ovulation, the transcription level of most AMP was higher (P < 0.05) compared with the luteal phase. Almost all candidate AMP mRNA expression was dependent on uterine health status, with a higher transcription level (P < 0.05) in inflamed endometrial tissues, especially during the late stage of the puerperium (Day 45-51 postpartum). Members of the DEFB family

  13. Serum amyloid A and pairing formyl peptide receptor 2 are expressed in corneas and involved in inflammation-mediated neovascularization

    PubMed Central

    Ren, Sheng-Wei; Qi, Xia; Jia, Chang-Kai; Wang, Yi-Qiang

    2014-01-01

    AIM To solidify the involvement of Saa-related pathway in corneal neovascularization (CorNV). The pathogenesis of inflammatory CorNV is not fully understood yet, and our previous study implicated that serum amyloid A (Saa) 1 (Saa1) and Saa3 were among the genes up-regulated upon CorNV induction in mice. METHODS Microarray data obtained during our profiling project on CorNV were analyzed for the genes encoding the four SAA family members (Saa1-4), six reported SAA receptors (formyl peptide receptor 2, Tlr2, Tlr4, Cd36, Scarb1, P2rx7) and seven matrix metallopeptidases (Mmp) 1a, 1b, 2, 3, 9, 10, 13 reportedly to be expressed upon SAA pathway activation. The baseline expression or changes of interested genes were further confirmed in animals with CorNV using molecular or histological methods. CorNV was induced in Balb/c and C57BL/6 mice by placing either three interrupted 10-0 sutures or a 2 mm filter paper soaked with sodium hydroxide in the central area of the cornea. At desired time points, the corneas were harvested for histology examination or for extraction of mRNA and protein. The mRNA levels of Saa1, Saa3, Fpr2, Mmp2 and Mmp3 in corneas were detected using quantitative reverse transcription-PCR, and SAA3 protein in tissues detected using immunohistochemistry or western blotting. RESULTS Microarray data analysis revealed that Saa1, Saa3, Fpr2, Mmp2, Mmp3 messengers were readily detected in normal corneas and significantly up-regulated upon CorNV induction. The changes of these five genes were confirmed with real-time PCR assay. On the contrary, other SAA members (Saa2, Saa4), other SAA receptors (Tlr2, Tlr4, Cd36, P2rx7, etc), or other Mmps (Mmp1a, Mmp1b, Mmp9, Mmp10, Mmp13) did not show consistent changes. Immunohistochemistry study and western blotting further confirmed the expression of SAA3 products in normal corneas as well as their up-regulation in corneas with CorNV. CONCLUSION SAA-FPR2 pathway composing genes were expressed in normal murine corneas and

  14. Expression of natriuretic peptides, nitric oxide synthase, and guanylate cyclase activity in frog mesonephros during the annual cycle.

    PubMed

    Fenoglio, Carla; Visai, Livia; Addario, Concetta; Gerzeli, Giuseppe; Milanesi, Gloria; Vaccarone, Rita; Barni, Sergio

    2004-06-01

    Natriuretic peptides (NPs), a family of structurally related hormones and nitric oxide (NO), generated by nitric oxide synthase (NOS), are believed to be involved in the regulation of fluid balance and sodium homeostasis. Differential expression and regulation of these factors depend on both physiological and pathological conditions. Both NPs and NO act in target organs through the activation of guanylate cyclase (GC) and the generation of guanosine 3',5'-cyclic monophosphate (cGMP), which is considered a common messenger for the action of these factors. The present study was designed to investigate--by histochemical methods--the expression of some NPs (proANP and ANP) and isoforms of NOS (neuronal NOS, nNOS, and inducible NOS, iNOS) in the mesonephros of Rana esculenta in different periods of the year including hibernation, to evaluate possible seasonal changes in their expression. We also studied the enzyme activity of NOS-related nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) and of GC. The experiments were performed on pieces of kidney of R. esculenta collected in their natural environment during active and hibernating life. The study was carried out using immunohistochemical techniques to demonstrate proANP, ANP, and some NOS isoforms. Antigen capture by enzyme linked immunosorbent assay (ELISA) was also performed to determine the presence of NPs in the frog kidney extract. Enzyme histochemistry was used to demonstrate the NOS-related NADPHd activity at light microscopy; GC activity was visualized at the electron microscope, using cerium as capture agent. The application of the immunohistochemical techniques demonstrated that frog mesonephros tubules express different patterns of distribution and/or expression of ANP and NOS during the annual cycle. Comparing the results obtained on active and hibernating frogs has provided interesting data; the NOS/NADPHd and GC activities showed some variations as well. Furthermore, the presence of NPs in

  15. Effects of Helicobacter pylori infection on gut appetite peptide (leptin, ghrelin) expression in elderly inpatients.

    PubMed

    Salles, Nathalie; Ménard, Armelle; Georges, Agnès; Salzmann, Mélanie; de Ledinghen, Victor; de Mascarel, Antoine; Emeriau, Jean-Paul; Lamouliatte, Hervé; Mégraud, Francis

    2006-11-01

    The aim of the study was to investigate the relationship between gastritis and leptin and ghrelin in elderly patients. Patients older than 75 years undergoing an endoscopy were included. We reported data on nutritional status and Helicobacter pylori infection diagnosis (serology, 13C-urea breath test, culture, histology, and polymerase chain reaction on gastric biopsies). Gastric messenger RNA expression of leptin and ghrelin were quantified by real-time polymerase chain reaction. Sixty-two patients were included (84.7 +/- 5.2 years). H. pylori infection was associated with decreased gastric expression of leptin (p = .021), ghrelin (p =.002), and plasma ghrelin levels (p = .018). Atrophy was associated with decreased gastric leptin (p = .007) and ghrelin (p = .02). H. pylori infection correlated negatively with patient energy intake (r = -0.36; p = .001) and body mass index (r = -0.34; p = .018). The negative association between ghrelin and H. pylori infection may be related to a higher prevalence of atrophy and raises the possibility that H. pylori may be contributing to undernutrition in some older people.

  16. cDNA sequence and expression analysis of an antimicrobial peptide, theromacin, in the triangle-shell pearl mussel Hyriopsis cumingii.

    PubMed

    Xu, Qiaoqing; Wang, Gailing; Yuan, Hanwen; Chai, Yi; Xiao, Zhili

    2010-09-01

    Bivalve molluscs rely on the interaction between cellular and humoral factors for protection against potential pathogens. Antimicrobial peptides (AMPs) have been proven to be one of the most important humoral components that afford resistance to pathogen infection. The AMP gene to be identified was that encoding theromacin in the triangle-shell pearl mussel Hyriopsis cumingii (Hc theromacin); this gene was identified from a suppression subtractive hybridization library, and subsequently cloned by 3' and 5' rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length theromacin cDNA contains 547 bp, with a 294-bp open reading frame that encodes a 97-amino acid peptide, and the deduced peptide sequence contains a 61-amino acid putative mature peptide. The sequence also contains 10 cysteine residues. Reverse transcriptase (RT)-PCR analysis showed that Hc theromacin transcripts were constitutively expressed in the liver, foot, gill, adductor muscle, heart, mantle, intestine, and hemocytes, with the highest level in hemocytes. Theromacin mRNA levels were found to increase after challenge with gram-positive and gram-negative bacteria. After injection of the gram-positive bacteria Staphylococcus aureus and Bifidobacterium bifidum, Hc theromacin expression showed the highest fold-change at 48 and 36 h after infection, respectively, and its levels decreased gradually thereafter. PMID:20639135

  17. Egg ovotransferrin‐derived ACE inhibitory peptide IRW increases ACE2 but decreases proinflammatory genes expression in mesenteric artery of spontaneously hypertensive rats

    PubMed Central

    Majumder, Kaustav; Liang, Guanxiang; Chen, Yanhong; Guan, LeLuo; Davidge, Sandra T.

    2015-01-01

    Scope Egg ovotransferrin‐derived angiotensin converting enzyme (ACE) inhibitory peptide IRW was previously shown to reduce blood pressure in spontaneously hypertensive rats through reduced vascular inflammation and increased nitric oxide‐mediated vasorelaxation. The main objective of the present study was to investigate the molecular mechanism of this peptide through transcriptome analysis by RNAseq technique. Methods and results Total RNA was extracted from kidney and mesenteric arteries; the RNAseq libraries (from untreated and IRW‐treated groups) were constructed and subjected to sequence using HiSeq 2000 system (Illumina) system. A total of 12 764 and 13 352 genes were detected in kidney and mesenteric arteries, respectively. The differentially expressed (DE) genes between untreated and IRW‐treated groups were identified and the functional analysis through ingenuity pathway analysis revealed a greater role of DE genes identified from mesenteric arteries than that of kidney in modulating various cardiovascular functions. Subsequent qPCR analysis further confirmed that IRW significantly increased the expression of ACE‐2, ABCB‐1, IRF‐8, and CDH‐1 while significantly decreased the expression ICAM‐1 and VCAM‐1 in mesenteric arteries. Conclusion Our research showed for the first time that ACE inhibitory peptide IRW could contribute to its antihypertensive activity through increased ACE2 and decreased proinflammatory genes expression. PMID:26016560

  18. Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection

    PubMed Central

    Spencer, John David; Jackson, Ashley R.; Li, Birong; Ching, Christina B.; Vonau, Martin; Easterling, Robert S.; Schwaderer, Andrew L.; McHugh, Kirk M.; Becknell, Brian

    2015-01-01

    Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain. PMID:26658437

  19. The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain

    SciTech Connect

    Burri, Dominique J.; Pasquato, Antonella; Ramos da Palma, Joel; Igonet, Sebastien; Oldstone, Michael B.A.; Kunz, Stefan

    2013-02-05

    Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.

  20. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    PubMed

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.

  1. Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection.

    PubMed

    Spencer, John David; Jackson, Ashley R; Li, Birong; Ching, Christina B; Vonau, Martin; Easterling, Robert S; Schwaderer, Andrew L; McHugh, Kirk M; Becknell, Brian

    2015-01-01

    Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain. PMID:26658437

  2. Staphylococcus aureus in the House Fly: Temporospatial Fate of Bacteria and Expression of the Antimicrobial Peptide defensin

    PubMed Central

    NAYDUCH, DANA; CHO, HANNAH; JOYNER, CHESTER

    2013-01-01

    House flies disseminate numerous species of bacteria acquired during feeding and breeding activities in microbe-rich habitats. Previous house fly surveys have detected the pathogen Staphylococcus aureus Rosenbach 1884, which causes cutaneous and septic infections in mammals, and enterotoxic food poisoning. We assessed the fate of GFP-expressing S. aureus (GFP-S. aureus) in the house fly alimentary canal with microscopy and by culture of whole flies and excreta. Furthermore, the concurrent expression of the antimicrobial peptide gene defensin was measured in the crop, proventriculus, midgut, and fat body. As soon as 4 h postingestion (PI), GFP-S. aureus were visualized as cocci or diplococci in the hindgut and rectum of flies fed ≈105 colony forming units. Bacteria persisted up to 6 h PI but significantly decreased. Excretion of viable GFP-S. aureus peaked at 2 h PI and, although significantly less, continued up to 4 h PI. defensin was highly upregulated locally in the alimentary canal and systemically in fat body at 2, 4, and 6 h PI making this study the first to report, to our knowledge, an epithelial and systemic response to a bacterium with lysine-type peptidoglycan in flies exposed via feeding. While flies harbored S. aureus for up to 6 h PI, the highest probability of vectoring biologically relevant amounts of bacteria occurred 0–2 h PI. The combined effects of excretion, digestion and antimicrobial effectors likely contribute to loss of ingested bacteria. Nonetheless, house flies are relevant vectors for S. aureus up to 2 h PI and environmental reservoirs up to 6 h PI. PMID:23427667

  3. 64Cu Labeled Sarcophagine Exendin-4 for MicroPET Imaging of Glucagon like Peptide-1 Receptor Expression

    PubMed Central

    Wu, Zhanhong; Liu, Shuanglong; Nair, Indu; Omori, Keiko; Scott, Stephen; Todorov, Ivan; Shively, John E.; Conti, Peter S.; Li, Zibo; Kandeel, Fouad

    2014-01-01

    The Glucagon-like peptide 1 receptor (GLP-1R) has become an important target for imaging due to its elevated expression profile in pancreatic islets, insulinoma, and the cardiovascular system. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), several studies have conjugated different chelators to a more stable analog of GLP-1 (such as exendin-4) as PET or SPECT imaging agents with various advantages and disadvantages. Based on the recently developed Sarcophagin chelator, here, we describe the construction of GLP-1R targeted PET probes containing monomeric and dimeric exendin-4 subunit. The in vitro binding affinity of BarMalSar-exendin-4 and Mal2Sar-(exendin-4)2 was evaluated in INS-1 cells, which over-express GLP-1R. Mal2Sar-(exendin-4)2 demonstrated around 3 times higher binding affinity compared with BaMalSar-exendin-4. After 64Cu labeling, microPET imaging of 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 were performed on subcutaneous INS-1 tumors, which were clearly visualized with both probes. The tumor uptake of 64Cu-Mal2Sar-(exendin-4)2 was significantly higher than that of 64Cu-BaMaSarl-exendin-4, which could be caused by polyvalency effect. The receptor specificity of these probes was confirmed by effective blocking of the uptake in both tumor and normal positive organs with 20-fold excess of unlabeled exendin-4. In conclusion, sarcophagine cage conjugated exendin-4 demonstrated persistent and specific uptake in INS-1 insulinoma model. Dimerization of exendin-4 could successfully lead to increased tumor uptake in vivo. Both 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 hold a great potential for GLP-1R targeted imaging. PMID:24955138

  4. Staphylococcus aureus in the house fly: temporospatial fate of bacteria and expression of the antimicrobial peptide defensin.

    PubMed

    Nayduch, Dana; Cho, Hannah; Joyner, Chester

    2013-01-01

    House flies disseminate numerous species of bacteria acquired during feeding and breeding activities in microbe-rich habitats. Previous house fly surveys have detected the pathogen Staphylococcus aureus Rosenbach 1884, which causes cutaneous and septic infections in mammals, and enterotoxic food poisoning. We assessed the fate of GFP-expressing S. aureus (GFP-S. aureus) in the house fly alimentary canal with microscopy and by culture of whole flies and excreta. Furthermore, the concurrent expression of the antimicrobial peptide gene defensin was measured in the crop, proventriculus, midgut, and fat body. As soon as 4 h postingestion (PI), GFP-S. aureus were visualized as cocci or diplococci in the hindgut and rectum of flies fed approximately equal 10(5) colony forming units. Bacteria persisted up to 6 h PI but significantly decreased. Excretion of viable GFP-S. aureus peaked at 2 h PI and, although significantly less, continued up to 4 h PI. defensin was highly upregulated locally in the alimentary canal and systemically in fat body at 2, 4, and 6 h PI making this study the first to report, to our knowledge, an epithelial and systemic response to a bacterium with lysine-type peptidoglycan in flies exposed via feeding. While flies harbored S. aureus for up to 6 h PI, the highest probability of vectoring biologically relevant amounts of bacteria occurred 0-2 h PI. The combined effects of excretion, digestion and antimicrobial effectors likely contribute to loss of ingested bacteria. Nonetheless, house flies are relevant vectors for S. aureus up to 2 h PI and environmental reservoirs up to 6 h PI.

  5. Cyclic mechanical stretch down-regulates cathelicidin antimicrobial peptide expression and activates a pro-inflammatory response in human bronchial epithelial cells.

    PubMed

    Karadottir, Harpa; Kulkarni, Nikhil Nitin; Gudjonsson, Thorarinn; Karason, Sigurbergur; Gudmundsson, Gudmundur Hrafn

    2015-01-01

    Mechanical ventilation (MV) of patients can cause damage to bronchoalveolar epithelium, leading to a sterile inflammatory response, infection and in severe cases sepsis. Limited knowledge is available on the effects of MV on the innate immune defense system in the human lung. In this study, we demonstrate that cyclic stretch of the human bronchial epithelial cell lines VA10 and BCi NS 1.1 leads to down-regulation of cathelicidin antimicrobial peptide (CAMP) gene expression. We show that treatment of VA10 cells with vitamin D3 and/or 4-phenyl butyric acid counteracted cyclic stretch mediated down-regulation of CAMP mRNA and protein expression (LL-37). Further, we observed an increase in pro-inflammatory responses in the VA10 cell line subjected to cyclic stretch. The mRNA expression of the genes encoding pro-inflammatory cytokines IL-8 and IL-1β was increased after cyclic stretching, where as a decrease in gene expression of chemokines IP-10 and RANTES was observed. Cyclic stretch enhanced oxidative stress in the VA10 cells. The mRNA expression of toll-like receptor (TLR) 3, TLR5 and TLR8 was reduced, while the gene expression of TLR2 was increased in VA10 cells after cyclic stretch. In conclusion, our in vitro results indicate that cyclic stretch may differentially modulate innate immunity by down-regulation of antimicrobial peptide expression and increase in pro-inflammatory responses. PMID:26664810

  6. Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study.

    PubMed

    Campbell, A P; Bautista, D L; Tripet, B; Wong, W Y; Irvin, R T; Hodges, R S; Sykes, B D

    1997-10-21

    The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has recently been the target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. We have successfully cloned and bacterially expressed a 15N-labeled PAK pilin peptide spanning residues 128-144 of the intact PAK pilin protein, PAK 128-144(Hs145), and have determined the solution secondary structure of this peptide using heteronuclear multidimensional NMR spectroscopy. The oxidized recombinant peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around the Ile138-Pro139 peptide bond. The pattern of NOEs, temperature coefficients, and coupling constants observed for the trans isomer demonstrate the presence of a type I beta-turn and a type II beta-turn spanning Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively. This is in agreement with the NMR solution structure of the trans isomer of a synthetic PAK 128-144 peptide which showed a type I and a type II beta-turn in these same regions of the sequence [McInnes, C., Sönnichsen, F. D., Kay, C. M., Hodges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hodges, R. S., and Sykes, B. D. (1995) Biochemistry 34, 16255-16268]. The pattern of NOEs, temperature coefficients, and coupling constants observed for the cis isomer also demonstrate a type II beta-turn spanning Pro139-Lys-Gly-Cys142, but suggest a second beta-turn spanning Asp132-Gln-Asp-Glu135. Thus, the cis isomer may also possess a double-turn motif (like the trans isomer), but with different spacing between the turns and a different placement of the first turn in the sequence. The discovery of a double-turn motif in the trans (and cis) recombinant PAK pilin peptide is an extremely important result since the double turn has been implicated as a structural requirement for the recognition of both receptor

  7. Effect of genetic SSTR4 ablation on inflammatory peptide and receptor expression in the non-inflamed and inflamed murine intestine.

    PubMed

    Van Op den Bosch, Joeri; Torfs, Pascal; De Winter, Benedicte Y; De Man, Joris G; Pelckmans, Paul A; Van Marck, Eric; Grundy, David; Van Nassauw, Luc; Timmermans, Jean-Pierre

    2009-09-01

    The recently suggested pivotal role of somatostatin (SOM) receptor 4 (SSTR4) in inflammation and nociception in several non-intestinal organs and in gastrointestinal (GI) physiology, necessitates exploration of the role of SSTR4 in GI pathophysiology. Therefore, the role of SSTR4 in GI activity was explored by investigating the effects of SSTR4 deficiency on intestinal motility, smooth muscle contractility and on the expression of SSTRs and neuropeptides in the healthy and Schistosoma mansoni-infected murine small intestine. Functional experiments revealed no differences in intestinal motility or smooth muscle cell contractility between wild-type and SSTR4 knockout (SSTR4(-/-)) mice in physiological conditions. As revealed by multiple immunofluorescent labellings, RT-PCR and quantitative real time RT-PCR (qPCR), genetic deficiency of SSTR4 considerably altered the expression of SOM and SSTRs in non-inflamed and inflamed conditions, affecting both extrinsic and intrinsic components of the intestinal innervation, along with SSTR expression in several non-neuronal cell types. Moreover, substance P and calcitonin gene-related peptide expression were significantly elevated in SSTR4(-/-) mice, confirming the modulatory role of SSTR4 on intestinal pro-inflammatory neuropeptide expression. These data suggest that SSTR4 plays a previously unexpected modulatory role in the regulation of intestinal SSTR expression. Moreover, in addition to the recently described inhibitory effects of SSTR4 on the neuronal release of pro-inflammatory peptides, SSTR4 appears also to be involved in the neuronal expression of both pro- and anti-inflammatory peptides in the murine small intestine.

  8. Antimicrobial peptides.

    PubMed

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  9. Effects of rizatriptan on the expression of calcitonin gene-related peptide and cholecystokinin in the periaqueductal gray of a rat migraine model.

    PubMed

    Yao, Gang; Han, Ximei; Hao, Tingting; Huang, Qian; Yu, Tingmin

    2015-02-01

    Triptans are serotonin 5-hydroxytryptamine receptor 1B/D agonists that are highly effective in the treatment of migraine. We previously found that rizatriptan can reduce the expression of proenkephalin and P substance in the rat midbrain, suggesting that rizatriptan may exert its analgesic effects by influencing the endogenous pain modulatory system. Calcitonin gene-related peptide (CGRP) and cholecystokinin (CCK) are mainly responsible for antagonizing the analgesic effects of opioid peptides in the endogenous pain modulatory system. In this study, we investigated the effects of rizatriptan on the expression of CGRP and CCK in the periaqueductal gray (PAG), a key structure of the endogenous pain modulatory system, in a rat migraine model induced by nitroglycerin. We found that the mRNA and protein levels of CGRP and CCK in the PAG of migraine rats were significantly increased compared to those in control rats, and these levels were significantly reduced upon treatment with rizatriptan in migraine rats (P<0.05). Our results suggest that the expression of CGRP and CCK in the endogenous pain modulatory system may be increased during migraine attacks, which further antagonizes the analgesic effects of endogenous opioid peptides and induces sustained migraine. Rizatriptan, however, significantly reduces the levels of CGRP and CCK to enhance the inhibition of pain signals via the endogenous pain modulatory system, resulting in effective treatment of migraine. PMID:25524408

  10. Radiohybridization PET imaging of KRAS G12D mRNA expression in human pancreas cancer xenografts with [(64)Cu]DO3A-peptide nucleic acid-peptide nanoparticles.

    PubMed

    Chakrabarti, A; Zhang, K; Aruva, M R; Cardi, C A; Opitz, A W; Wagner, N J; Thakur, M L; Wickstrom, E

    2007-06-01

    There is a compelling need to image pancreas cancer at an early stage. Human pancreas cancer cells display elevated levels of KRAS protein due to high copy numbers of KRAS mRNA, and elevated levels of insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. Therefore we hypothesized that pancreas cancer could be detected in vivo with a single probe that targets both KRAS mRNA and IGF1R. Because positron emission tomography (PET) is a sensitive imaging technique, we designed a probe incorporating the positron-emitting nuclide (64)Cu. The KRAS-specific hybridization probe consisted of 1,4,7-tris(carboxymethylaza)cyclododecane-10-aza-acetyl (DO3A) on the N-terminus of a peptide nucleic acid (PNA) hybridization sequence (GCCATCAGCTCC) linked to a cyclized IGF1 peptide analog (d-Cys-Ser-Lys-Cys) on the C-terminus, for IGF1R-mediated endocytosis. A series of such KRAS radiohybridization probes with 0, 1, 2 or 3 mismatches to KRAS G12D mRNA, including exact matches to wild type KRAS mRNA and KRAS G12V mRNA, along with a double d(Ala) replacement IGF1 peptide control, were assembled by continuous solid phase synthesis. To test the hypothesis that KRAS-IGF1 dual probes could specifically image KRAS mRNA expression noninvasively in human IGF1R-overexpressing AsPC1 pancreas cancer xenografts in immunocompromised mice, [(64)Cu]PNA radiohybridization probes and controls were administered by tail vein. The [(64)Cu]KRAS-IGF1 radiohybridization probe yielded strong tumor contrast in PET images, 8.6 +/- 1.4-fold more intense in the center of human pancreas cancer xenografts than in the contralateral muscle at 4 h post-injection. Control experiments with single base KRASmismatches, an IGF1 peptide mismatch, and a breast cancer xenograft lacking KRAS activation yielded weak tumor contrast images. These experiments are consistent with our hypothesis for noninvasive PET imaging of KRAS oncogene expression in pancreas cancer xenografts. Imaging oncogene m

  11. Anti-microbial peptide (AMP): nucleotide variation, gene expression, and host resistance in the white pine blister rust (WPBR) pathosystem.

    PubMed

    Liu, Jun-Jun; Zamany, Arezoo; Sniezko, Richard A

    2013-01-01

    Pinus monticola antimicrobial peptide (PmAMP1) inhibits growth of Cronartium ribicola and other fungal pathogens. C. ribicola causes white pine blister rust and has resulted in a dramatic reduction of native white pines across North America. Quantitative disease resistance (QDR) is a highly desirable trait screened in breeding programs for durable resistance against C. ribicola. Along with phenotyping on a collection of germplasms, we analyzed PmAMP1 transcript and protein expression and re-sequenced the full-length gene including its promoter region. A mixed linear model was used to identify the association of single nucleotide polymorphisms (SNPs) with accumulated protein and stem QDR levels. Among 16 PmAMP1 SNPs identified in the present study, we found an association of protein levels with 6 SNPs (P < 0.05), including 2 in the 5'-untranslated region (UTR), 3 in the open reading frame (ORF) region with 2 nonsynonymous SNPs, and 1 SNP in the 3'-UTR. Another set of six SNPs was associated with stem QDR levels (P < 0.05), with one localized in the promoter region and the other five in the ORF region with four nonsynonymous changes, suggesting that multiple isoforms may have antifungal activity to differing degrees. Of three common PmAMP1 haplotypes, the trees with haplotype 2 showed high QDR levels with moderate protein abundance while those trees with haplotype 3 exhibited low QDR levels in the susceptible range and the lowest level of protein accumulation. Thus, an association of gene variations with protein abundance and resistance-related traits may facilitate elucidation of physiological contribution of PmAMP1 to host resistance.

  12. Identification of HLA-A24-binding peptides of Mycobacterium tuberculosis derived proteins with beta 2m linked HLA-A24 single chain expressing cells.

    PubMed

    Ding, Jie; Wang, Yan; Cheng, Tingting; Chen, Xiaowei; Gao, Bin

    2010-01-01

    Tuberculosis is caused by an intracellular pathogen Mycobacterium tuberculosis (Mtb) and poses a persistent threat to global health. MHC class I-restricted CD8 cytotoxic T lymphocytes (CTL) are essential for protective immunity to Tuberculosis. Information for CTL epitopes derived from Mtb is desirable for vaccine design and assessment of T cell responses. However, the knowledge about CTL epitopes of Mtb, particularly those non-A2 HLA alleles restricted is rare. In this study, beta-2-microglobulin (beta 2m, beta(2)m) linked HLA-A24 single chain was expressed on RMA-S cell line defective in the endogenous antigen processing and applied for screening of peptides which could stabilize the HLA-A24 complex on the cell surface. From a group of peptides predicted as binders by a computer algorithm, five peptides were shown to bind to HLA-A24 protein on the cell surface. As comparison we have also identified a dozen Mtb proteins derived peptides that bind to HLA-A2 specifically. The cell line and HLA binders present here would be useful for further identification of CD8 restricted Mtb epitopes.

  13. Molecular cloning and functional expression of a chicken intestinal peptide transporter (cPepT1) in Xenopus oocytes and Chinese hamster ovary cells.

    PubMed

    Chen, Hong; Pan, YuanXiang; Wong, Eric A; Bloomquist, Jeffrey R; Webb, Kenneth E

    2002-03-01

    To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken duodenal cDNA library. The cDNA was 2914 bp long and encoded a protein of 714 amino acid residues with an estimated molecular size of 79.3 kDa and an isoelectric point of 7.48. cPepT1 protein is similar60% identical to PepT1 from rabbits, humans, mice, rats and sheep. Sixteen dipeptides, three tripeptides and four tetrapeptides that contained the essential amino acids Met, Lys and(or) Trp were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary cells. For most di- and tripeptides tested, the substrate affinities were in the micromolar range, indicating that cPepT1 has high affinity for these peptides. Lys-Lys and Lys-Trp-Lys were exceptions, with substrate affinities in the millimolar range. Neither free amino acids nor tetrapeptides were transported by cPepT1. Northern blot analysis using a full-length cPepT1 cDNA as the probe demonstrated that cPepT1 is expressed strongly in the duodenum, jejunum and ileum, and at lower levels in kidney and ceca. The present study demonstrated for the first time the presence and functional characteristics of a peptide transport system from an avian species.

  14. A LuxR homolog in a cottonwood tree endophyte that activates gene expression in response to a plant signal or specific peptides

    DOE PAGES

    Schaefer, Amy L.; Oda, Yasuhiro; Coutinho, Bruna Goncalves; Pelletier, Dale A.; Weiburg, Justin; Venturi, Vittorio; Greenberg, E. Peter; Harwood, Caroline S.

    2016-08-02

    Homologs of the LuxR acyl-homoserine lactone (AHL) quorum-sensing signal receptor are prevalent in Proteobacteria isolated from roots of the Eastern cottonwood tree, Populus deltoides. Many of these isolates possess an orphan LuxR homolog, closely related to OryR from the rice pathogen Xanthomonas oryzae. OryR does not respond to AHL signals but, instead, responds to an unknown plant compound. We discovered an OryR homolog, PipR, in the cottonwood endophyte Pseudomonas sp. strain GM79. The genes adjacent to pipR encode a predicted ATP-binding cassette (ABC) peptide transporter and peptidases. We purified the putative peptidases, PipA and AapA, and confirmed their predicted activities.more » A transcriptional pipA-gfp reporter was responsive to PipR in the presence of plant leaf macerates, but it was not influenced by AHLs, similar to findings with OryR. We found that PipR also responded to protein hydrolysates to activate pipA-gfp expression. Among many peptides tested, the tripeptide Ser-His-Ser showed inducer activity but at relatively high concentrations. An ABC peptide transporter mutant failed to respond to leaf macerates, peptone, or Ser-His-Ser, while peptidase mutants expressed higher-than-wild-type levels of pipA-gfp in response to any of these signals. Our studies are consistent with a model where active transport of a peptidelike signal is required for the signal to interact with PipR, which then activates peptidase gene expression. As a result, the identification of a peptide ligand for PipR sets the stage to identify plant-derived signals for the OryR family of orphan LuxR proteins.« less

  15. A LuxR Homolog in a Cottonwood Tree Endophyte That Activates Gene Expression in Response to a Plant Signal or Specific Peptides

    PubMed Central

    Schaefer, Amy L.; Oda, Yasuhiro; Coutinho, Bruna Goncalves; Pelletier, Dale A.; Weiburg, Justin; Venturi, Vittorio; Greenberg, E. Peter

    2016-01-01

    ABSTRACT Homologs of the LuxR acyl-homoserine lactone (AHL) quorum-sensing signal receptor are prevalent in Proteobacteria isolated from roots of the Eastern cottonwood tree, Populus deltoides. Many of these isolates possess an orphan LuxR homolog, closely related to OryR from the rice pathogen Xanthomonas oryzae. OryR does not respond to AHL signals but, instead, responds to an unknown plant compound. We discovered an OryR homolog, PipR, in the cottonwood endophyte Pseudomonas sp. strain GM79. The genes adjacent to pipR encode a predicted ATP-binding cassette (ABC) peptide transporter and peptidases. We purified the putative peptidases, PipA and AapA, and confirmed their predicted activities. A transcriptional pipA-gfp reporter was responsive to PipR in the presence of plant leaf macerates, but it was not influenced by AHLs, similar to findings with OryR. We found that PipR also responded to protein hydrolysates to activate pipA-gfp expression. Among many peptides tested, the tripeptide Ser-His-Ser showed inducer activity but at relatively high concentrations. An ABC peptide transporter mutant failed to respond to leaf macerates, peptone, or Ser-His-Ser, while peptidase mutants expressed higher-than-wild-type levels of pipA-gfp in response to any of these signals. Our studies are consistent with a model where active transport of a peptidelike signal is required for the signal to interact with PipR, which then activates peptidase gene expression. The identification of a peptide ligand for PipR sets the stage to identify plant-derived signals for the OryR family of orphan LuxR proteins. PMID:27486195

  16. Satiation and Stress-Induced Hypophagia: Examining the Role of Hindbrain Neurons Expressing Prolactin-Releasing Peptide or Glucagon-Like Peptide 1

    PubMed Central

    Maniscalco, James W.; Kreisler, Alison D.; Rinaman, Linda

    2013-01-01

    Neural circuits distributed within the brainstem, hypothalamus, and limbic forebrain interact to control food intake and energy balance under normal day-to-day conditions, and in response to stressful conditions under which homeostasis is threatened. Experimental studies using rats and mice have generated a voluminous literature regarding the functional organization of circuits that inhibit food intake in response to satiety signals, and in response to stress. Although the central neural bases of satiation and stress-induced hypophagia often are studied and discussed as if they were distinct, we propose that both behavioral states are generated, at least in part, by recruitment of two separate but intermingled groups of caudal hindbrain neurons. One group comprises a subpopulation of noradrenergic (NA) neurons within the caudal nucleus of the solitary tract (cNST; A2 cell group) that is immunopositive for prolactin-releasing peptide (PrRP). The second group comprises non-adrenergic neurons within the cNST and nearby reticular formation that synthesize glucagon-like peptide 1 (GLP-1). Axonal projections from PrRP and GLP-1 neurons target distributed brainstem and forebrain regions that shape behavioral, autonomic, and endocrine responses to actual or anticipated homeostatic challenge, including the challenge of food intake. Evidence reviewed in this article supports the view that hindbrain PrRP and GLP-1 neurons contribute importantly to satiation and stress-induced hypophagia by modulating the activity of caudal brainstem circuits that control food intake. Hindbrain PrRP and GLP-1 neurons also engage hypothalamic and limbic forebrain networks that drive parallel behavioral and endocrine functions related to food intake and homeostatic challenge, and modulate conditioned and motivational aspects of food intake. PMID:23346044

  17. Significant expression of a Chinese scorpion peptide, BmK1, in Escherichia coli through promoter engineering and gene dosage strategy.

    PubMed

    Wang, Jianfeng; Xiong, Zhiqiang; Yang, Yingying; Zhao, Na; Wang, Yong

    2014-01-01

    Heterologous expression is an efficient alternative to conventional extraction to produce a specific Buthus martensii Karsch (BmK) peptide. In this work, BmK1 was successfully expressed in Escherichia coli after genetic codon optimization, but BmK1 content was <6% of total cellular protein. To improve BmK1 expression, a trc promoter library with a wide relative strength was constructed, and three promoters, PpJF136 (0.55), PpJF325 (1.29), and PpJF288 (2.31), were selected to control BmK1 expression. A higher BmK1 expression (>13.9% of total protein) was obtained using a stronger promoter, PpJF325 . Furthermore, a maximum BmK1 content (>21.7% of total protein) was obtained by combining promoter PpJF325 and three copies of the BmK1 gene. The yield of the purified BmK1 achieved 196.74 mg L(-1) in E. coli BL21(DE3) pJF431, which was improved 2.09-fold compared with the control. This was the highest reported production of scorpion peptides in E. coli. PMID:24372571

  18. Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide.

    PubMed

    Raemaekers, R J; de Muro, L; Gatehouse, J A; Fordham-Skelton, A P

    1999-10-01

    Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces alpha-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the alpha-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the alpha-factor prosequence. Polypeptides in which most of the alpha-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the alpha-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.

  19. Neurokinin B-related Peptide Suppresses the Expression of GnRH I, Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus.

    PubMed

    Jin, Ye Hwa; Park, Jin Woo; Kim, Jung-Hyun; Kwon, Joon Yeong

    2016-03-01

    Neurokinin B (NKB) and neurokinin B related peptide (NKBRP) belong to tachykinin peptide family. Theyact as a neurotransmitter and/or neuromodulator. Mutation of NKB and/or its cognate receptor, NK3R resulted in hypogonadotropic hypogonadism in mammals, implying a strong involvement of NKB/NK3R system in controlling mammalian reproduction. Teleosts possess NKBRP as well as NKB, but their roles in fish reproduction need to be clarified. In this study, NKB and NKBRP coding gene (tac3) was cloned from Nile tilapia and sequenced. Based on the sequence, Nile tilapia NKB and NKBRP peptide were synthesized and their biological potencies were tested in vitro pituitary culture. The synthetic NKBRP showed direct inhibitory effect on the expression of GTH subunits at the pituitary level. This inhibitory effect was confirmed in vivo by means of intraperitoneal (ip) injection of synthetic NKB and NKBRP to mature female tilapia (20 pmol/g body weight [BW]). Both NKB and NKBRP had no effect on the plasma level of sex steroids, E2 and 11-KT. However, NKBRP caused declines of expression level of GnRH I, Kiss2 and tac3 mRNAs in the brain while NKB seemed to have no distinct effect. These results indicate some inhibitory roles of NKBRP in reproduction of mature female Nile tilapia, although their exact functions are not clear at the moment. PMID:27294210

  20. Recombinant expression of porcine lactoferrin peptide LF-6 with intein technology and its immunomodulatory function in ETEC K88-infected mice.

    PubMed

    Jiang, Qin; Zhang, Haiwen; Xie, Yonggang; Wang, Yizhen

    2016-10-01

    LF-6 is a modified antibacterial peptide derived from LFP-20, a major active ingredient of porcine lactoferrin, whose antibacterial activity is 200 times higher than its native protein counterpart. Moreover, LF-6 displays even higher antibacterial activity than LFP-20 and negligible toxic adverse effects, make it a potential therapeutic agent for antibacterial purposes. Escherichia coli expression system has been a preferred choice and workhorse for most recombinant proteins. However, LF-6 must be coexpressed with a fusion partner to avoid its potentially fatal toxicity which would threat E. coli expression system. In this study, we successfully introduced intein system to solve this problem, which LF-6 was N-terminally fused to dithiothreitol (DTT)-induced self-cleavable intein, and it conduct cleavage when the intein-fusion peptide passing through a chromatography column filled with chitin, then the spliced peptide was purified with RP-HPLC and identified with mass spectroscopy. A bacteriostatic test showed that the recombinant LF-6 displayed nearly the same antibacterial activity as the chemically synthetized LF-6, and an in vivo immunoprotection analysis showed that the recombinant LF-6 exerted protective effects on Escherichia coli (ETEC)-K88-infected mice, which significantly reduced the pro-inflammatory cytokines level in plasma and intestine, and resistant to intestinal mucosal injury compared to the infective alone groups. Our study indicates that the intein system allows a safe and efficient method to produce recombinant LF-6, which not only has antibacterial activity, but more importantly, has an immunomodulatory function.

  1. Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system.

    PubMed

    Zhang, Xu; Lian, Jiazhang; Kai, Lei; Huang, Lei; Cen, Peilin; Xu, Zhinan

    2014-02-01

    Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10⁻¹⁴ cm³ s⁻¹ monomer⁻¹. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve. PMID:24411442

  2. Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system.

    PubMed

    Zhang, Xu; Lian, Jiazhang; Kai, Lei; Huang, Lei; Cen, Peilin; Xu, Zhinan

    2014-02-01

    Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10⁻¹⁴ cm³ s⁻¹ monomer⁻¹. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve.

  3. Expression of conformationally constrained adhesion peptide in an antibody CDR loop and inhibition of natural killer cell cytotoxic activity by an antibody antigenized with the RGD motif.

    PubMed Central

    Zanetti, M; Filaci, G; Lee, R H; del Guercio, P; Rossi, F; Bacchetta, R; Stevenson, F; Barnaba, V; Billetta, R

    1993-01-01

    We report that an antibody engineered to express three Arg-Gly-Asp (RGD) repeats in the third complementarity-determining region of the heavy chain (antigenized antibody) efficiently inhibits the lysis of human erythroleukemia K-562 cells by natural killer (NK) cells. Synthetic peptides containing RGD did not inhibit. Inhibition was specific for the (RGD)3-containing loop and required simultaneous occupancy of the Fc receptor (CD16) on effector cells. The antigenized antibody inhibited other forms of cytotoxicity mediated by NK cells but not cytotoxicity mediated by major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL). A three-dimensional model of the engineered antibody loop shows the structure and physicochemical characteristics probably required for the ligand activity. The results indicate that an RGD motif is involved in the productive interaction between NK and target cells. Moreover, they show that peptide expression in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. This is a new approach towards imparting ligand properties to antibody molecules and can be used to study the biological function and specificity of short peptide motifs, including those involved in cell adhesion. Images PMID:8223447

  4. Antimicrobial Peptides in Reptiles

    PubMed Central

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  5. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions.

  6. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions. PMID:26374891

  7. Expression, purification, crystallization and preliminary crystallographic analysis of hepatitis B virus core protein dimerized via a peptide linker containing an EGFP insertion.

    PubMed

    Kikuchi, Masaki; Iwabuchi, Shinichiro; Kikkou, Tatsuhiko; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi; Kawata, Masaaki; Sato, Chikara; Matsumoto, Osamu

    2013-08-01

    Virus-like particles (VLPs) have many potentially useful applications. The core proteins of human hepatitis B virus self-assemble into icosahedral VLPs. As previously reported, core protein dimers (CPDs), produced by connecting two core proteins via a peptide linker, can also assemble into VLPs. CPDs in which heterologous proteins were connected to the C-terminus (CPD1) were found to rearrange into symmetrical octahedra during crystallization. In this study, a heterologous protein was inserted into the peptide linker of the CPD (CPD2). CPD2 was expressed in Escherichia coli, assembled into VLPs, purified and crystallized. A single crystal diffracted to 2.8 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 218.6 Å. Single-crystal analysis showed that CPD1 and CPD2 rearranged into the same octahedral organization in a crystallization solution.

  8. Differential expression of IGF-I mRNA and peptide in the male and female gonad during early development of a bony fish, the tilapia Oreochromis niloticus.

    PubMed

    Berishvili, Giorgi; D'Cotta, Helena; Baroiller, Jean-François; Segner, Helmut; Reinecke, Manfred

    2006-05-01

    Insulin-like growth factor I (IGF-I) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. The major source of circulating IGF-I is liver, but IGF-I-producing cells also occur in other organs, including the gonads. Because no data are available on the potential production sites of IGF-I in gonad development, developmental stages of monosex breedings of male and female tilapia from 0 day postfertilization (DPF) to 90 DPF were investigated for the production sites of IGF-I at the peptide (immunohistochemistry) and mRNA (in situ hybridization) level. IGF-I mRNA first appeared in somatic cells of the male and female gonad anlage at 7 DPF followed by IGF-I peptide around 9-10 DPF. Gonad anlagen were detected from 7 DPF. Starting at 7 DPF, IGF-I peptide but no IGF-I mRNA was observed in male and female primordial germ cells (PGCs) provided that IGF-I mRNA was not under the detection level, this observation may suggest that IGF-I originates from the somatic cells and is transferred to the PGCs or is of maternal origin. While in female germ cells IGF-I mRNA and peptide appeared at 29 DPF, in male germ cells both were detected as late as at 51-53 DPF. It is assumed that the production of IGF-I in the germ cells is linked to the onset of meiosis that in tilapia ovary starts at around 28 DPF and in testes at around 52-53 DPF. In adult testis, IGF-I mRNA and peptide occurred in the majority of spermatogonia and spermatocytes as well as in Leydig cells, the latter indicating a role of IGF-I in the synthesis of male sex steroids. In adult ovary, IGF-I mRNA and IGF-I peptide were always present in small and previtellogenic oocytes but only IGF-I peptide infrequently occurred in oocytes at the later stages. IGF-I expression appeared in numerous granulosa and some theca cells of follicles at the lipid stage and persisted in follicles with mature oocytes. The results suggest a crucial role of local IGF-I in the formation

  9. Antimicrobial Peptides from Fish

    PubMed Central

    Masso-Silva, Jorge A.; Diamond, Gill

    2014-01-01

    Antimicrobial peptides (AMPs) are found widely distributed through Nature, and participate in the innate host defense of each species. Fish are a great source of these peptides, as they express all of the major classes of AMPs, including defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins. As with other species, the fish peptides exhibit broad-spectrum antimicrobial activity, killing both fish and human pathogens. They are also immunomodulatory, and their genes are highly responsive to microbes and innate immuno-stimulatory molecules. Recent research has demonstrated that some of the unique properties of fish peptides, including their ability to act even in very high salt concentrations, make them good potential targets for development as therapeutic antimicrobials. Further, the stimulation of their gene expression by exogenous factors could be useful in preventing pathogenic microbes in aquaculture. PMID:24594555

  10. Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites.

    PubMed

    Husseneder, Claudia; Donaldson, Jennifer R; Foil, Lane D

    2016-01-01

    The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.

  11. Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites

    PubMed Central

    Husseneder, Claudia; Donaldson, Jennifer R.; Foil, Lane D.

    2016-01-01

    The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival. PMID:26985663

  12. Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites.

    PubMed

    Husseneder, Claudia; Donaldson, Jennifer R; Foil, Lane D

    2016-01-01

    The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival. PMID:26985663

  13. Specific expression of GFP{sub uv}-{beta}1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide

    SciTech Connect

    Kato, Tatsuya; Park, Enoch Y. . E-mail: yspark@agr.shizuoka.ac.jp

    2007-08-03

    Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFP{sub uv}-{beta}1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP{sup -} ) and its bacmid. The secretion efficiencies of all signal peptides were 15-30% in Bm5 cells and 24-30% in silkworm larvae, while that of the +16 signal peptide was 0% in Bm5 cells and 1% in silkworm larvae. The fusion protein that contained the +16 signal peptide was expressed specifically in the endoplasmic reticulum (ER) and in the fractions of cell precipitations. Ninety-four percent of total intracellular {beta}1,3-N-acetylglucosaminyltransferase ({beta}3GnT) activity was detected in cell precipitations following the 600, 8000, and 114,000g centrifugations. In the case of the +38 signal peptide, 60% of total intracellular activity was detected in the supernatant from the 114,000g spin, and only 1% was found in the precipitate. Our results suggest that the +16 signal peptide might be situated in the transmembrane region and not cleaved by signal peptidase in silkworm or B. mori cells. Therefore, the fusion protein connected to the +16 signal peptide stayed in the fat body of silkworm larvae with biological function, and was not secreted extracellularly.

  14. Vasoactive intestinal peptide/vasoactive intestinal peptide receptor relative expression in salivary glands as one endogenous modulator of acinar cell apoptosis in a murine model of Sjögren's syndrome.

    PubMed

    Hauk, V; Calafat, M; Larocca, L; Fraccaroli, L; Grasso, E; Ramhorst, R; Leirós, C Pérez

    2011-12-01

    Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by a progressive oral and ocular dryness that correlates poorly with the autoimmune damage of the glands. It has been proposed that a loss of homeostatic equilibrium in the glands is partly responsible for salivary dysfunction with acinar cells involved actively in the pathogenesis of SS. The non-obese diabetic (NOD) mouse model of Sjögren's syndrome develops secretory dysfunction and early loss of glandular homeostatic mechanisms, with mild infiltration of the glands. Based on the vasodilator, prosecretory and trophic effects of the vasoactive intestinal peptide (VIP) on acini as well as its anti-inflammatory properties we hypothesized that the local expression of VIP/vasoactive intestinal peptide receptor (VPAC) system in salivary glands could have a role in acinar cell apoptosis and macrophage function thus influencing gland homeostasis. Here we show a progressive decline of VIP expression in submandibular glands of NOD mice with no changes in VPAC receptor expression compared with normal mice. The deep loss of endogenous VIP was associated with a loss of acinar cells through apoptotic mechanisms that could be induced further by tumour necrosis factor (TNF)-α and reversed by VIP through a cyclic adenosine-5'-monophosphate (cAMP)/protein kinase A (PKA)-mediated pathway. The clearance of apoptotic acinar cells by macrophages was impaired for NOD macrophages but a shift from inflammatory to regulatory phenotype was induced in macrophages during phagocytosis of apoptotic acinar cells. These results support that the decline in endogenous VIP/VPAC local levels might influence the survival/apoptosis intracellular set point in NOD acinar cells and their clearance, thus contributing to gland homeostasis loss.

  15. Atrial natriuretic peptide inhibits the expression of tissue factor and plasminogen activator inhibitor 1 induced by angiotensin II in cultured rat aortic endothelial cells.

    PubMed

    Yoshizumi, M; Tsuji, H; Nishimura, H; Kasahara, T; Sugano, T; Masuda, H; Nakagawa, K; Nakahara, Y; Kitamura, H; Yamada, K; Yoneda, M; Sawada, S; Nakagawa, M

    1998-03-01

    The pharmacological characteristics of atrial natriuretic peptide (ANP), such as natriuresis, vasodilation, or suppression of smooth muscle cell proliferation, are well investigated. However, this is the first study to report its role on blood coagulation and fibrinolysis mediated by vascular endothelial cells. In this study, the effects of ANP on the enhanced expression of tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) by angiotensin II (Ang II) in cultured rat aortic endothelial cells (RAECs) were examined. The expressions of TF and PAI-1 mRNA were detected by northern blotting methods. The activities of TF on the surface of RAECs and PAI-1 in the culture media were measured by chromogenic assay. ANP suppressed mRNA expressions of TF and PAI-1 induced by Ang II in a concentration-dependent manner. This suppression was accompanied by the decreased activities of TF and PAI-1.

  16. Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans.

    PubMed

    Cadenas, R F; Gil, J A; Martín, J F

    1992-12-01

    The alpha-amylase gene (amy) from Streptomyces griseus IMRU 3570 and the beta-galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P2 or P3. The alpha-amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum.

  17. Expression analyses of insulin-like peptide 3, RXFP2, LH receptor, and 3β-hydroxysteroid dehydrogenase in testes of normal and cryptorchid dogs.

    PubMed

    Hannan, M A; Kawate, N; Kubo, Y; Pathirana, I N; Büllesbach, E E; Hatoya, S; Inaba, T; Takahashi, M; Tamada, H

    2015-10-15

    Insulin-like peptide 3 (INSL3) plays a key role in testicular descent in rodents, whereas in domestic animals, many aspects of the roles of INSL3 in reproductive organs after puberty are still unknown. This study was undertaken to (1) determine the quantitative changes of gene expression of testicular INSL3, its receptor (RXFP2), LH receptor, and 3β-hydroxysteroid dehydrogenase during and after puberty in normal male dogs; (2) compare the expressions of these substances in normal and cryptorchid dogs; and (3) localize the cells expressing INSL3 in normal and retained canine testes. Testes were obtained from small-breed normal male dogs (n = 56) and cryptorchid dogs (n = 22). Normal scrotal testes from the normal dogs (normal testes), retained testes from both the unilateral and bilateral cryptorchid dogs (retained testes), and scrotal testes of the unilateral cryptorchid dogs (cryptorchid scrotal testes) were used. We measured the concentrations of these testicular messenger RNAs (mRNAs) by quantitative real-time reverse transcription polymerase chain reaction, and an enzyme immunoassay was used for measuring INSL3 peptide. Immunohistochemistry for INSL3 peptide was done in paraformaldehyde-fixed frozen testicular tissue. In the normal dogs, total amount of INSL3 mRNA per testis tended to decrease (P = 0.05) from pubertal (6-12 months) to postpubertal (1-5 years) and decreased (P < 0.01) to middle age (5-10 years), but total amount of INSL3 peptide per testis did not change among age groups. Concentrations of INSL3 mRNA were higher (P < 0.01) in retained testes than those in the normal testes and cryptorchid scrotal testes, and similar differences were observed for INSL3 peptide. Reversely, total amounts of INSL3 mRNA and peptide per retained testis were lower (P < 0.01) than those per normal testis because of smaller weight of retained testes. Concentrations and total amount of RXFP2 mRNA in the retained testes were almost nil and lower (P < 0.01) than those in

  18. Amelioration of Cardiac Function and Activation of Anti-Inflammatory Vasoactive Peptides Expression in the Rat Myocardium by Low Level Laser Therapy

    PubMed Central

    Manchini, Martha Trindade; Serra, Andrey Jorge; Feliciano, Regiane dos Santos; Santana, Eduardo Tadeu; Antônio, Ednei Luis; de Tarso Camillo de Carvalho, Paulo; Montemor, Jairo; Crajoinas, Renato Oliveira; Girardi, Adriana Castello Costa; Tucci, Paulo José Ferreira; Silva, José Antônio

    2014-01-01

    Low-level laser therapy (LLLT) has been used as an anti-inflammatory treatment in several disease conditions, even when inflammation is a secondary consequence, such as in myocardial infarction (MI). However, the mechanism by which LLLT is able to protect the remaining myocardium remains unclear. The present study tested the hypothesis that LLLT reduces inflammation after acute MI in female rats and ameliorates cardiac function. The potential participation of the Renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS) vasoactive peptides was also evaluated. LLLT treatment effectively reduced MI size, attenuated the systolic dysfunction after MI, and decreased the myocardial mRNA expression of interleukin-1 beta and interleukin-6 in comparison to the non-irradiated rat tissue. In addition, LLLT treatment increased protein and mRNA levels of the Mas receptor, the mRNA expression of kinin B2 receptors and the circulating levels of plasma kallikrein compared to non-treated post-MI rats. On the other hand, the kinin B1 receptor mRNA expression decreased after LLLT. No significant changes were found in the expression of vascular endothelial growth factor (VEGF) in the myocardial remote area between laser-irradiated and non-irradiated post-MI rats. Capillaries density also remained similar between these two experimental groups. The mRNA expression of the inducible nitric oxide synthase (iNOS) was increased three days after MI, however, this effect was blunted by LLLT. Moreover, endothelial NOS mRNA content increased after LLLT. Plasma nitric oxide metabolites (NOx) concentration was increased three days after MI in non-treated rats and increased even further by LLLT treatment. Our data suggest that LLLT diminishes the acute inflammation in the myocardium, reduces infarct size and attenuates left ventricle dysfunction post-MI and increases vasoactive peptides expression and nitric oxide (NO) generation. PMID:24991808

  19. Amelioration of cardiac function and activation of anti-inflammatory vasoactive peptides expression in the rat myocardium by low level laser therapy.

    PubMed

    Manchini, Martha Trindade; Serra, Andrey Jorge; Feliciano, Regiane dos Santos; Santana, Eduardo Tadeu; Antônio, Ednei Luis; de Tarso Camillo de Carvalho, Paulo; Montemor, Jairo; Crajoinas, Renato Oliveira; Girardi, Adriana Castello Costa; Tucci, Paulo José Ferreira; Silva, José Antônio

    2014-01-01

    Low-level laser therapy (LLLT) has been used as an anti-inflammatory treatment in several disease conditions, even when inflammation is a secondary consequence, such as in myocardial infarction (MI). However, the mechanism by which LLLT is able to protect the remaining myocardium remains unclear. The present study tested the hypothesis that LLLT reduces inflammation after acute MI in female rats and ameliorates cardiac function. The potential participation of the Renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS) vasoactive peptides was also evaluated. LLLT treatment effectively reduced MI size, attenuated the systolic dysfunction after MI, and decreased the myocardial mRNA expression of interleukin-1 beta and interleukin-6 in comparison to the non-irradiated rat tissue. In addition, LLLT treatment increased protein and mRNA levels of the Mas receptor, the mRNA expression of kinin B2 receptors and the circulating levels of plasma kallikrein compared to non-treated post-MI rats. On the other hand, the kinin B1 receptor mRNA expression decreased after LLLT. No significant changes were found in the expression of vascular endothelial growth factor (VEGF) in the myocardial remote area between laser-irradiated and non-irradiated post-MI rats. Capillaries density also remained similar between these two experimental groups. The mRNA expression of the inducible nitric oxide synthase (iNOS) was increased three days after MI, however, this effect was blunted by LLLT. Moreover, endothelial NOS mRNA content increased after LLLT. Plasma nitric oxide metabolites (NOx) concentration was increased three days after MI in non-treated rats and increased even further by LLLT treatment. Our data suggest that LLLT diminishes the acute inflammation in the myocardium, reduces infarct size and attenuates left ventricle dysfunction post-MI and increases vasoactive peptides expression and nitric oxide (NO) generation. PMID:24991808

  20. Synthesis, expression and purification of a type of chlorotoxin-like peptide from the scorpion, Buthus martensii Karsch, and its acute toxicity analysis.

    PubMed

    Fu, Yue-jun; Yin, Li-tian; Wang, Wei; Chai, Bao-feng; Liang, Ai-hua

    2005-10-01

    A gene, rBmK Cta, encoding a chlorotoxin-like peptide from the scorpion, Buthus martensii Karsch, was synthesized according to the sequence optimized for codon usage in Escherichia coli and was expressed in E. coli BL21 (DE3) using a pExSecI expression system in which the IgG-binding domain-ZZ of protein A is fused to the N-terminal of rBmK CTa. The fusion protein, ZZ-rBmK CTa, was expressed in soluble form (7.8 mg l(-1)) and was purified to give a single band on SDS-PAGE. The domain-ZZ of fusion protein ZZ-rBmK CTa was removed by cleavage of an Asn-Gly peptide bond with hydroxylamine. The rBmK CTa was separated from the IgG-binding moiety by a second passage through the IgG affinity column. Western blot analysis demonstrated that this protein was rBmK CTa. Acute toxicity assay in mice demonstrated that the rBmK CTa had an LD(50) value of 4.3 mg kg(-1).

  1. Extracellular expression of YlLip11 with a native signal peptide from Yarrowia lipolytica MSR80 in three different yeast hosts.

    PubMed

    Kumari, Arti; Baronian, Keith; Kunze, Gotthard; Gupta, Rani

    2015-06-01

    Lipase YlLip11 from Yarrowia lipolytica was expressed with a signal peptide encoding sequence in Arxula adeninivorans, Saccharomyces cerevisiae and Hansenula polymorpha using the Xplor®2 transformation/expression platform and an expression module with the constitutive Arxula-derived TEF1 promoter. The YlLip11 signal peptide was functional in all of the yeast hosts with 97% of the recombinant enzyme being secreted into the culture medium. However, recombinant YlLip11 with His Tag fused at C-terminal was not active. The best recombinant YlLip11 producing A. adeninivorans G1212/YRC102-YlLip11 transformant cultivated in shake flasks produced 2654 U/L lipase, followed by S. cerevisiae SEY6210/YRC103-YlLip11 (1632U/L) and H. polymorpha RB11/YRC103-YlLip11 (1144U/L). Although the biochemical parameters of YlLip11 synthesized in different hosts were similar, their glycosylation level and thermo stability differed. The protein synthesized by the H. polymorpha transformant had the highest degree of glycosylation and with a t1/2 of 60min at 70°C, exhibited the highest thermostability. PMID:25725269

  2. Peptide nanotubes.

    PubMed

    Hamley, Ian W

    2014-07-01

    The self-assembly of different classes of peptide, including cyclic peptides, amyloid peptides and surfactant-like peptides into nanotube structures is reviewed. The modes of self-assembly are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized.

  3. Modulation of chicken intestinal immune gene expression by small cationic peptides as feed additives during the first week posthatch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have been investigating modulation strategies tailored around the selective stimulation of the host’s immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soi...

  4. 35S Promoter Methylation in Kanamycin-Resistant Kalanchoe (Kalanchoe pinnata L.) Plants Expressing the Antimicrobial Peptide Cecropin P1 Transgene.

    PubMed

    Shevchuk, T V; Zakharchenko, N S; Tarlachkov, S V; Furs, O V; Dyachenko, O V; Buryanov, Y I

    2016-09-01

    Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases. PMID:27682168

  5. Serpinin: A Novel Chromogranin A-Derived, Secreted Peptide Up-Regulates Protease Nexin-1 Expression and Granule Biogenesis in Endocrine Cells

    PubMed Central

    Koshimizu, Hisatsugu; Cawley, Niamh X.; Kim, Taeyoon; Yergey, Alfred L.

    2011-01-01

    Previously we demonstrated that chromogranin A (CgA) promoted secretory granule biogenesis in endocrine cells by stabilizing and preventing granule protein degradation in the Golgi, through up-regulation of expression of the protease inhibitor, protease nexin-1 (PN-1). However, the mechanism by which CgA signals the increase of PN-1 expression is unknown. Here we identified a 2.9-kDa CgA-C-terminus peptide, which we named serpinin, in conditioned media from AtT-20 cells, a corticotroph cell line, which up-regulated PN-1 mRNA expression. Serpinin was secreted from AtT-20 cells upon high potassium stimulation and increased PN-1 mRNA transcription in these cells, in an actinomycin D-inhibitable manner. CgA itself and other CgA-derived peptides, when added to AtT-20 cell media, had no effect on PN-1 expression. Treatment of AtT-20 cells with 10 nm serpinin elevated cAMP levels and PN-1 mRNA expression, and this effect was inhibited by a protein kinase A inhibitor, 6–22 amide. Serpinin and a cAMP analog, 8-bromo-cAMP, promoted the translocation of the transcription factor Sp1 into the nucleus, which is known to drive PN-1 expression. Additionally, an Sp1 inhibitor, mithramycin A inhibited the serpinin-induced PN-1 mRNA up-regulation. Furthermore, a luciferase reporter assay demonstrated serpinin-induced up-regulation of PN-1 promoter activity in an Sp1-dependent manner. When added to CgB-transfected 6T3 cells, a mutant AtT20 cell line, serpinin induced granule biogenesis as evidenced by the presence of CgB puncta accumulation in the processes and tips. Our findings taken together show that serpinin, a novel CgA-derived peptide, is secreted upon stimulation of corticotrophs and plays an important autocrine role in up-regulating PN-1-dependent granule biogenesis via a cAMP-protein kinase A-Sp1 pathway to replenish released granules. PMID:21436258

  6. Response of the gypsy moth, Lymantria dispar to transgenic poplar, Populus simonii x P. nigra, expressing fusion protein gene of the spider insecticidal peptide and Bt-toxin C-peptide.

    PubMed

    Cao, Chuan-Wang; Liu, Gui-Feng; Wang, Zhi-Ying; Yan, Shan-Chun; Ma, Ling; Yang, Chuan-Ping

    2010-01-01

    The response of the Asian gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) to a fusion gene consisting of the spider, Atrax robustus Simon (Araneae: Hexanthelidae) ω-ACTX-Ar1 sequence coding for an ω-atracotoxin and a sequence coding for the Bt-toxin C-peptide, expressed in transgenic poplar Populus simonii x P. nigra L. (Malphigiales: Salicaceae) was investigated. Individual performance, feeding selection, midgut proteinase activity and nutrition utilization were monitored. The growth and development of L. dispar were significantly affected by continually feeding on the transgenic poplar, with the larval instars displaying significantly shorter developmental times than those fed on nontransgenic poplar, but pupation was delayed. Mortality was higher in populations fed transgenic poplar leaves, than for larvae fed nontransgenic poplar leaves. The cumulative mortality during all stages of larvae fed transgenic leaves was 92% compared to 16.7% of larvae on nontransgenic leaves. The highest mortality observed was 71.7% in the last larval instar stage. A two-choice test showed that fifth-instar larvae preferred to feed on nontransgenic leaves at a ratio of 1:1.4. Feeding on transgenic leaves had highly significant negative effects on relative growth of larvae, and the efficiency of conversion of ingested and digested food. Activity of major midgut proteinases was measured using substrates TAME and BTEE showed significant increases in tryptase and chymotrypsinlike activity (9.2- and 9.0-fold, respectively) in fifth-instar larvae fed on transgenic leaves over control. These results suggest transgenic poplar is resistant to L. dispar, and the mature L. dispar may be weakened by the transgenic plants due to Bt protoxins activated by elevated major midgut proteinase activity. The new transgenic poplar expressing fusion protein genes of Bt and a new spider insecticidal peptide are good candidates for managing gypsy moth.

  7. Short chain fatty acids (propionic and hexanoic) decrease Staphylococcus aureus internalization into bovine mammary epithelial cells and modulate antimicrobial peptide expression.

    PubMed

    Alva-Murillo, Nayeli; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

    2012-03-23

    Short chain fatty acids (SCFAs) are critical nutrients for ruminants and are mainly obtained from bacterial fermentation of carbohydrates. In addition to their nutrimental function, SCFAs have antimicrobial and anti-inflammatory properties, as well as immunomodulatory roles. It has been reported that sodium butyrate reduces Staphylococcus aureus internalization into bovine mammary epithelial cells (bMEC) and modulates antimicrobial peptide mRNA expression. Nevertheless, it has not been evaluated if sodium propionate (NaP) and sodium hexanoate (NaH) have similar actions. Since they are present in milk, the aim of this study was to determinate the effect of both SCFAs on S. aureus internalization into bMEC and to evaluate their effects on modulation of innate immunity elements. Our data showed that both SCFAs (0.25-5mM) did not affect S. aureus growth and bMEC viability. By gentamicin protection assay (MOI 30:1) we showed that NaP and NaH reduced bacterial internalization into bMEC, which ranged 27-55% and 39-65%, respectively, in relation to non treated controls. Also, both SCFAs up-regulate tracheal antimicrobial peptide (TAP) mRNA expression; however, bovine neutrophil β-defensin 5 (BNBD5) mRNA expression was not modified or was down-regulated. In addition, TAP and BNBD5 expression was up-regulated by S. aureus. Finally, the decrease in bacterial internalization under SCFA treatments is not related to nitric oxide production. In conclusion, NaP and NaH decrease S. aureus internalization into bMEC and modulate TAP gene expression, which may be related to the reduction in bacterial internalization. PMID:21930351

  8. Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine

    PubMed Central

    Gutierrez-Mecinas, Maria; Polgár, Erika; Todd, Andrew J

    2016-01-01

    Background Gastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells. Results Injection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP–EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP–EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK. Conclusions Both expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway. PMID:27270268

  9. A novel vertebrates Toll-like receptor counterpart regulating the anti-microbial peptides expression in the freshwater crayfish, Procambarus clarkii.

    PubMed

    Wang, Zheng; Chen, Yi-Hong; Dai, Yun-Jia; Tan, Jing-Min; Huang, Ying; Lan, Jiang-Feng; Ren, Qian

    2015-03-01

    Toll-like receptors (TLRs) play an important role in regulation of anti-microbial peptides (AMPs) expression. A novel vertebrates TLR counterpart named PcToll, was firstly identified from the freshwater crayfish, Procambarus clarkii. Phylogenetic analysis showed that PcToll together with Drosophila melanogaster and Anopheles gambiae Toll9 were clustered with human Tolls. PcToll was mainly expressed in hepatopancreas and gills and it also could be detected in hemocytes, heart, stomach and intestine. PcToll was upregulated in hemocytes and gills post 24 h Vibrio anguillarum challenge. In hepatopancreas and intestine, the highest expression level of PcToll could be observed at 12 h V. anguillarum challenge. In hemocytes, PcToll went up post 24 h Staphylococcus aureus challenge and in gills, the expression level of PcToll showed no obvious change from 2 to 24 h S. aureus challenge. In hepatopancreas post 12 h S. aureus challenge, PcToll was upregulated and it showed obvious upregulation post 12 h S. aureus challenge in intestine. RNAi results showed that PcToll was involved in regulation of crustins (Cru1, Cru2), anti-lipopolysaccharide factor 2 (ALF2) and lysozyme 1 (Lys1) expression. Overexpression of PcToll in Drosophila S2 cells could induce Drosophila Attacin (Atta), Metchnikowin (Mtk), Drosomycin (Drs) and shrimp Penaeidin (PEN4) expression. From the results, it could be speculated that PcToll might play important roles in crayfish innate immune defense.

  10. [Codon optimization and eukaryotic expression analysis of the analgesic peptide gene BmK AngM1 from Buthus martensii Karsch].

    PubMed

    Yang, Jin-ling; Gao, Li-li; Zhu, Ping; Hou, Qi; Wang, Fen; Yu, Wen-bo; Nie, Tao

    2012-10-01

    Codon bias is an important factor which influences heterologous gene expression. Optimizing codon sequence could improve expression level of heterologous gene. In order to improve the expression level of BmK AngM1 gene encoding the analgesic peptide from Buthus martensii Karsch in Pichia pastoris, the codon-optimized BmK AngM1 gene according to its cDNA sequence and the preference codon usage of P. pastoris were cloned into expression vector pPIC9K and then transformed into P. pastoris. The expersion of recombinant BmK AngM1 (rBmK AngM1) was inducced by methanol in the medium, and the expression level of the optimized BmK AngM1 gene was 3.7 times of the native one. These results suggested that the expression of BmK AngM1 in P. pastoris could be successfully improved by codon optimization. PMID:23289154

  11. Identification and analysis of drug-responsive expression of UDP-glucuronosyltransferase family 1 (UGT1) isozyme in rat hepatic microsomes using anti-peptide antibodies.

    PubMed

    Ikushiro, S; Emi, Y; Iyanagi, T

    1995-12-20

    Expression of rat hepatic UDP-glucuronosyltransferase family 1 (UGT1) isozymes has been examined using anti-peptide antibodies raised against a conserved carboxyl-terminal portion of all isozymes and variable amino-terminal portions of each isozyme of the phenol cluster (UGT1A) and bilirubin cluster (UGT1B). Among the isozymes expressed in rat hepatic microsomes, UGT1B1 (54 kDa) of bilirubin cluster was found to be a major form and minor forms were identified as UGT1A1 (53 kDa), UGT1B2 (56 kDa), and UGT1B5 (57 kDa). Using a combination of 2D sodium dodecyl sulfate gel electrophoresis and immunoblotting, all the isozymes were found to be simultaneously lacked in Gunn rat hepatic microsomes. The effects of various drugs as inducer on the expression of each UGT1 isozyme were analyzed. The UGT1A1 and UGT1A2 of the phenol cluster isozymes were significantly induced in 3-methylcholanthrene-treated rats. The expression of UGT1B1 and the glucuronidation activity toward bilirubin in rat hepatic microsomes were induced two- to threefold by clofibrate and dexamethasone administration. On the other hand, the regulation of UGT1B2 and UGT1B5 expression was different from that of UGT1B1. These results clearly show the drug-responsive expression of each UGT1 isozyme using isozyme-specific antibodies for the first time. PMID:8554318

  12. Recombinant expression of porcine lactoferrin peptide LF-6 with intein technology and its immunomodulatory function in ETEC K88-infected mice.

    PubMed

    Jiang, Qin; Zhang, Haiwen; Xie, Yonggang; Wang, Yizhen

    2016-10-01

    LF-6 is a modified antibacterial peptide derived from LFP-20, a major active ingredient of porcine lactoferrin, whose antibacterial activity is 200 times higher than its native protein counterpart. Moreover, LF-6 displays even higher antibacterial activity than LFP-20 and negligible toxic adverse effects, make it a potential therapeutic agent for antibacterial purposes. Escherichia coli expression system has been a preferred choice and workhorse for most recombinant proteins. However, LF-6 must be coexpressed with a fusion partner to avoid its potentially fatal toxicity which would threat E. coli expression system. In this study, we successfully introduced intein system to solve this problem, which LF-6 was N-terminally fused to dithiothreitol (DTT)-induced self-cleavable intein, and it conduct cleavage when the intein-fusion peptide passing through a chromatography column filled with chitin, then the spliced peptide was purified with RP-HPLC and identified with mass spectroscopy. A bacteriostatic test showed that the recombinant LF-6 displayed nearly the same antibacterial activity as the chemically synthetized LF-6, and an in vivo immunoprotection analysis showed that the recombinant LF-6 exerted protective effects on Escherichia coli (ETEC)-K88-infected mice, which significantly reduced the pro-inflammatory cytokines level in plasma and intestine, and resistant to intestinal mucosal injury compared to the infective alone groups. Our study indicates that the intein system allows a safe and efficient method to produce recombinant LF-6, which not only has antibacterial activity, but more importantly, has an immunomodulatory function. PMID:27487204

  13. A Family of CSαβ Defensins and Defensin-Like Peptides from the Migratory Locust, Locusta migratoria, and Their Expression Dynamics during Mycosis and Nosemosis

    PubMed Central

    Zhang, Liwei; Zhang, Pengfei; Zhang, Long

    2016-01-01

    Insect defensins are effector components of the innate defense system. During infection, these peptides may play a role in the control of pathogens by providing protective antimicrobial barriers between epithelial cells and the hemocoel. The cDNAs encoding four defensins of the migratory locust, Locusta migratoria, designated LmDEF 1, 3–5, were identified for the first time by transcriptome-targeted analysis. Three of the members of this CSαβ defensin family, LmDEF 1, 3, and 5, were detected in locust tissues. The pro regions of their sequences have little-shared identities with other insect defensins, though the predicted mature peptides align well with other insect defensins. Phylogenetic analysis indicates a completely novel position of both LmDEF 1 and 3, compared to defensins from hymenopterans. The expression patterns of the genes encoding LmDEFs in the fat body and salivary glands were studied in response to immune-challenge by the microsporidian pathogen Nosema locustae and the fungus Metarhizium anisopliae after feeding or topical application, respectively. Focusing on Nosema-induced immunity, qRT-PCR was employed to quantify the transcript levels of LmDEFs. A higher transcript abundance of LmDEF5 was distributed more or less uniformly throughout the fat body along time. A very low baseline transcription of both LmDEFs 1 and 3 in naïve insects was indicated, and that transcription increases with time or is latent in the fat body or salivary glands of infected nymphs. In the salivary glands, expression of LmDEF3 was 20-40-times higher than in the fat body post-microbial infection. A very low expression of LmDEF3 could be detected in the fat body, but eventually increased with time up to a maximum at day 15. Delayed induction of transcription of these peptides in the fat body and salivary glands 5–15 days post-activation and the differential expression patterns suggest that the fat body/salivary glands of this species are active in the immune response

  14. A Family of CSαβ Defensins and Defensin-Like Peptides from the Migratory Locust, Locusta migratoria, and Their Expression Dynamics during Mycosis and Nosemosis.

    PubMed

    Lv, Mingyue; Mohamed, Amr Ahmed; Zhang, Liwei; Zhang, Pengfei; Zhang, Long

    2016-01-01

    Insect defensins are effector components of the innate defense system. During infection, these peptides may play a role in the control of pathogens by providing protective antimicrobial barriers between epithelial cells and the hemocoel. The cDNAs encoding four defensins of the migratory locust, Locusta migratoria, designated LmDEF 1, 3-5, were identified for the first time by transcriptome-targeted analysis. Three of the members of this CSαβ defensin family, LmDEF 1, 3, and 5, were detected in locust tissues. The pro regions of their sequences have little-shared identities with other insect defensins, though the predicted mature peptides align well with other insect defensins. Phylogenetic analysis indicates a completely novel position of both LmDEF 1 and 3, compared to defensins from hymenopterans. The expression patterns of the genes encoding LmDEFs in the fat body and salivary glands were studied in response to immune-challenge by the microsporidian pathogen Nosema locustae and the fungus Metarhizium anisopliae after feeding or topical application, respectively. Focusing on Nosema-induced immunity, qRT-PCR was employed to quantify the transcript levels of LmDEFs. A higher transcript abundance of LmDEF5 was distributed more or less uniformly throughout the fat body along time. A very low baseline transcription of both LmDEFs 1 and 3 in naïve insects was indicated, and that transcription increases with time or is latent in the fat body or salivary glands of infected nymphs. In the salivary glands, expression of LmDEF3 was 20-40-times higher than in the fat body post-microbial infection. A very low expression of LmDEF3 could be detected in the fat body, but eventually increased with time up to a maximum at day 15. Delayed induction of transcription of these peptides in the fat body and salivary glands 5-15 days post-activation and the differential expression patterns suggest that the fat body/salivary glands of this species are active in the immune response

  15. Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

    PubMed Central

    Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G

    1997-01-01

    Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875

  16. Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

    PubMed

    Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G

    1997-07-01

    Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875

  17. Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis

    PubMed Central

    Bao, Xiaofeng; Pachikara, Niseema D.; Oey, Christopher B.; Balakrishnan, Amit; Westblade, Lars F.; Tan, Ming; Chase, Theodore; Nickels, Bryce E.

    2011-01-01

    Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability. PMID:21719536

  18. Wuschel-related homeobox5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation.

    PubMed

    Osipova, Maria A; Mortier, Virginie; Demchenko, Kirill N; Tsyganov, Victor E; Tikhonovich, Igor A; Lutova, Ludmila A; Dolgikh, Elena A; Goormachtig, Sofie

    2012-03-01

    In legumes, the symbiotic nodules are formed as a result of dedifferentiation and reactivation of cortical root cells. A shoot-acting receptor complex, similar to the Arabidopsis (Arabidopsis thaliana) CLAVATA1 (CLV1)/CLV2 receptor, regulating development of the shoot apical meristem, is involved in autoregulation of nodulation (AON), a mechanism that systemically controls nodule number. The targets of CLV1/CLV2 in the shoot apical meristem, the WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family transcription factors, have been proposed to be important regulators of apical meristem maintenance and to be expressed in apical meristem "organizers." Here, we focus on the role of the WOX5 transcription factor upon nodulation in Medicago truncatula and pea (Pisum sativum) that form indeterminate nodules. Analysis of temporal WOX5 expression during nodulation with quantitative reverse transcription-polymerase chain reaction and promoter-reporter fusion revealed that the WOX5 gene was expressed during nodule organogenesis, suggesting that WOX genes are common regulators of cell proliferation in different systems. Furthermore, in nodules of supernodulating mutants, defective in AON, WOX5 expression was higher than that in wild-type nodules. Hence, a conserved WUS/WOX-CLV regulatory system might control cell proliferation and differentiation not only in the root and shoot apical meristems but also in nodule meristems. In addition, the link between nodule-derived CLE peptides activating AON in different legumes and components of the AON system was investigated. We demonstrate that the identified AON component, NODULATION3 of pea, might act downstream from or beside the CLE peptides during AON. PMID:22232385

  19. Analysis of expression, cellular localization, and function of three inhibitors of apoptosis (IAPs) from Litopenaeus vannamei during WSSV infection and in regulation of antimicrobial peptide genes (AMPs).

    PubMed

    Wang, Pei-Hui; Wan, Ding-Hui; Gu, Zhi-Hua; Qiu, Wei; Chen, Yong-Gui; Weng, Shao-Ping; Yu, Xiao-Qiang; He, Jian-Guo

    2013-01-01

    Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei. LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrioalginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V. alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibriopenaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together

  20. Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

    PubMed Central

    Burck, P J; Berg, D H; Luk, T P; Sassmannshausen, L M; Wakulchik, M; Smith, D P; Hsiung, H M; Becker, G W; Gibson, W; Villarreal, E C

    1994-01-01

    The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein

  1. The antiarrhythmic peptide rotigaptide (ZP123) increases gap junction intercellular communication in cardiac myocytes and HeLa cells expressing connexin 43

    PubMed Central

    Clarke, Thomas C; Thomas, Dafydd; Petersen, Jørgen S; Evans, W Howard; Martin, Patricia E M

    2006-01-01

    We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL-1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43-GFP, Cx32-GFP, Cx26-GFP, wild-type Cx43 or wild-type Cx26. Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication-modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32-GFP, Cx26-GFP or wild-type Cx26. In contrast, HeLa cells expressing Cx43-GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication. Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL-1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes. Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed. We conclude that the effects of rotigaptide were confined to cells expressing Cx43. PMID:16415913

  2. Suppressor of cytokine signaling 2 (SOCS2) negatively regulates the expression of antimicrobial peptides by affecting the Stat transcriptional activity in shrimp Marsupenaeus japonicus.

    PubMed

    Sun, Jie-Jie; Lan, Jiang-Feng; Xu, Ji-Dong; Niu, Guo-Juan; Wang, Jin-Xing

    2016-09-01

    The suppressor of cytokine signaling (SOCS) family is a kind of negative regulators in the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in mammals and Drosophila. In kuruma shrimp, Marsupenaeus japonicus, SOCS2 is identified and its expression can be stimulated by peptidoglycan and polycytidylic acid. However, if SOCS2 participates in regulating Jak/Stat pathway in shrimp still needs further study. In this study, SOCS2 with Src homology 2 domain and SOCS box was identified in kuruma shrimp, M. japonicus. SOCS2 existed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine, the expression of SOCS2 was upregulated significantly in the hemocytes and intestine of shrimp challenged with Vibrio anguillarum at 6 h. To analyze SOCS2 function in shrimp immunity, bacterial clearance and survival rate were analyzed after knockdown of SOCS2 in shrimp challenged with V. anguillarum. Results showed that bacterial clearance increased, and the survival rate improved significantly comparing with controls. The SOCS2 was expressed in Escherichia coli and the recombinant SOCS2 was injected into shrimp, and Stat phosphorylation and translocation were analyzed. The result showed that "overexpression" of SOCS2 declined Stat phosphorylation level and inhibited Stat translocation into the nucleus. After knockdown of SOCS2 in shrimp prior to V. anguillarum infection, the expression level of antimicrobial peptides, including anti-lipopolysaccharide factors C1, C2 and D1, and Crustin I was upregulated significantly, and the expression of the AMPs was declined after recombinant SOCS2 injection. The SOCS2 expression was also decreased in Stat-knockdown shrimp challenged by V. anguillarum at 6 and 12 h. Therefore, SOCS2 negatively regulates the AMP expression by inhibiting Stat phosphorylation and translocation into nucleus in shrimp, meanwhile, SOCS2 expression was also regulated by Jak/Stat pathway.

  3. Suppressor of cytokine signaling 2 (SOCS2) negatively regulates the expression of antimicrobial peptides by affecting the Stat transcriptional activity in shrimp Marsupenaeus japonicus.

    PubMed

    Sun, Jie-Jie; Lan, Jiang-Feng; Xu, Ji-Dong; Niu, Guo-Juan; Wang, Jin-Xing

    2016-09-01

    The suppressor of cytokine signaling (SOCS) family is a kind of negative regulators in the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in mammals and Drosophila. In kuruma shrimp, Marsupenaeus japonicus, SOCS2 is identified and its expression can be stimulated by peptidoglycan and polycytidylic acid. However, if SOCS2 participates in regulating Jak/Stat pathway in shrimp still needs further study. In this study, SOCS2 with Src homology 2 domain and SOCS box was identified in kuruma shrimp, M. japonicus. SOCS2 existed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine, the expression of SOCS2 was upregulated significantly in the hemocytes and intestine of shrimp challenged with Vibrio anguillarum at 6 h. To analyze SOCS2 function in shrimp immunity, bacterial clearance and survival rate were analyzed after knockdown of SOCS2 in shrimp challenged with V. anguillarum. Results showed that bacterial clearance increased, and the survival rate improved significantly comparing with controls. The SOCS2 was expressed in Escherichia coli and the recombinant SOCS2 was injected into shrimp, and Stat phosphorylation and translocation were analyzed. The result showed that "overexpression" of SOCS2 declined Stat phosphorylation level and inhibited Stat translocation into the nucleus. After knockdown of SOCS2 in shrimp prior to V. anguillarum infection, the expression level of antimicrobial peptides, including anti-lipopolysaccharide factors C1, C2 and D1, and Crustin I was upregulated significantly, and the expression of the AMPs was declined after recombinant SOCS2 injection. The SOCS2 expression was also decreased in Stat-knockdown shrimp challenged by V. anguillarum at 6 and 12 h. Therefore, SOCS2 negatively regulates the AMP expression by inhibiting Stat phosphorylation and translocation into nucleus in shrimp, meanwhile, SOCS2 expression was also regulated by Jak/Stat pathway. PMID:27492125

  4. Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50▿

    PubMed Central

    Basanta, Antonio; Gómez-Sala, Beatriz; Sánchez, Jorge; Diep, Dzung B.; Herranz, Carmen; Hernández, Pablo E.; Cintas, Luis M.

    2010-01-01

    In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast. PMID:20348300

  5. Streptavidin-binding peptides and uses thereof

    NASA Technical Reports Server (NTRS)

    Szostak, Jack W. (Inventor); Wilson, David S. (Inventor); Keefe, Anthony D. (Inventor)

    2006-01-01

    The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest.

  6. Streptavidin-binding peptides and uses thereof

    NASA Technical Reports Server (NTRS)

    Szostak, Jack W. (Inventor); Wilson, David S. (Inventor); Keefe, Anthony D. (Inventor)

    2005-01-01

    The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest.

  7. Antimicrobial peptides

    PubMed Central

    2014-01-01

    With increasing antibiotics resistance, there is an urgent need for novel infection therapeutics. Since antimicrobial peptides provide opportunities for this, identification and optimization of such peptides have attracted much interest during recent years. Here, a brief overview of antimicrobial peptides is provided, with focus placed on how selected hydrophobic modifications of antimicrobial peptides can be employed to combat also more demanding pathogens, including multi-resistant strains, without conferring unacceptable toxicity. PMID:24758244

  8. Hypothalamic distribution, adenohypophyseal receptor expression, and ligand functionality of RFamide-related peptide 3 in the mare during the breeding and nonbreeding seasons.

    PubMed

    Thorson, Jennifer F; Prezotto, Ligia D; Cardoso, Rodolfo C; Sharpton, Sarah M; Edwards, John F; Welsh, Thomas H; Riggs, Penny K; Caraty, Alain; Amstalden, Marcel; Williams, Gary L

    2014-02-01

    RFamide-related peptide 3 (RFRP3), the mammalian homologue of avian gonadotropin-inhibitory hormone, has been shown to negatively regulate the secretion of LH and may contribute to reproductive seasonality in some species. Herein, we examined the presence and potential role of the RFRP3-signaling system in regulating LH secretion in the mare during the breeding and nonbreeding seasons. Hypothalamic NPVF mRNA (the precursor mRNA for RFRP3) was detected at the level of the dorsomedial nucleus and paraventricular nucleus, but expression did not change with season. A greater number of RFRP3-expressing cells was observed throughout the rostral-caudal extension of the dorsomedial nucleus. Furthermore, adenohypophyseal expression of the RFRP3 receptor (NPFFR1) during the winter anovulatory season did not differ from that during either the follicular or luteal phases of the estrous cycle. When tested in primary adenohypophyseal cell culture or in vivo during both the breeding and nonbreeding seasons, neither equine nor ovine peptide sequences for RFRP3 suppressed basal or GnRH-mediated release of LH. However, infusion of RF9, an RFRP3 receptor-signaling antagonist, into seasonally anovulatory mares induced a robust increase in secretion of LH both before and following continuous treatment with GnRH. The results indicate that the cellular machinery associated with RFRP3 function is present in the equine hypothalamus and adenohypophysis. However, evidence for functionality of the RFRP3-signaling network was only obvious when an antagonist RF9 was employed. Because GnRH-induced release of LH was not affected by RF9, its actions may occur upstream from the gonadotrope to stimulate or disinhibit secretion of GnRH.

  9. Expression of familial Alzheimer disease presenilin 1 gene attenuates vesicle traffic and reduces peptide secretion in cultured astrocytes devoid of pathologic tissue environment.

    PubMed

    Stenovec, Matjaž; Trkov, Saša; Lasič, Eva; Terzieva, Slavica; Kreft, Marko; Rodríguez Arellano, José Julio; Parpura, Vladimir; Verkhratsky, Alexei; Zorec, Robert

    2016-02-01

    In the brain, astrocytes provide metabolic and trophic support to neurones. Failure in executing astroglial homeostatic functions may contribute to the initiation and propagation of diseases, including Alzheimer disease (AD), characterized by a progressive loss of neurones over years. Here, we examined whether astrocytes from a mice model of AD isolated in the presymptomatic phase of the disease exhibit alterations in vesicle traffic, vesicular peptide release and purinergic calcium signaling. In cultured astrocytes isolated from a newborn wild-type (wt) and 3xTg-AD mouse, secretory vesicles and acidic endosomes/lysosomes were labeled by transfection with plasmid encoding atrial natriuretic peptide tagged with mutant green fluorescent protein (ANP.emd) and by LysoTracker, respectively. The intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored with Fluo-2 and visualized by confocal microscopy. In comparison with controls, spontaneous mobility of ANP- and LysoTracker-labeled vesicles was diminished in 3xTg-AD astrocytes; the track length (TL), maximal displacement (MD) and directionality index (DI) were all reduced in peptidergic vesicles and in endosomes/lysosomes (P < 0.001), as was the ATP-evoked attenuation of vesicle mobility. Similar impairment of peptidergic vesicle trafficking was observed in wt rat astrocytes transfected to express mutated presenilin 1 (PS1M146V). The ATP-evoked ANP discharge from single vesicles was less efficient in 3xTg-AD and PS1M146V-expressing astrocytes than in respective wt controls (P < 0.05). Purinergic stimulation evoked biphasic and oscillatory [Ca(2+)]i responses; the latter were less frequent (P < 0.001) in 3xTg-AD astrocytes. Expression of PS1M146V in astrocytes impairs vesicle dynamics and reduces evoked secretion of the signaling molecule ANP; both may contribute to the development of AD.

  10. Molecular cloning, characterization and mRNA expression of two antibacterial peptides: crustin and anti-lipopolysaccharide factor in swimming crab Portunus trituberculatus.

    PubMed

    Yue, Feng; Pan, Luqing; Miao, Jingjing; Zhang, Lin; Li, Jian

    2010-06-01

    In this study, the cDNAs of a crustin and an anti-lipopolysaccharide factor (ALF) were cloned from the hemocytes of swimming crab Portunus trituberculatus using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of P. trituberculatus crustin (denoted as PtCrustin) was of 584 bp, including an open reading frame (ORF) of 333 bp encoding a 21 amino acids signal peptide and a mature polypeptide of 89 amino acids with the predicted molecular mass of 10.08 kDa. The typical conserved cysteine residues containing WAP domain at the C-terminal displayed high similarity to other crustins. Moreover, the full-length cDNA of P. trituberculatus ALF (denoted as PtALF) was of 1050 bp, including an ORF of 372 bp encoding a 26 amino acids signal peptide and a mature polypeptide of 97 amino acids with the predicted molecular mass of 11.35 kDa. PtALF contained two conserved cysteine residues and showed high similarity to other crab ALFs. Using fluorescent real-time quantitative PCR, PtCrustin mRNA was expressed at the highest level in hemocytes and higher level in stomach while extremely low in hepatopancreas, muscle, gill and heart. In contrast, the PtALF mRNA expression was observed in all tissues. The relative expression levels of PtCrustin and PtALF in hemocytes of P. trituberculatus showed a clear time-dependent response after challenge with Vibrio alginolyticus. These results indicate that PtCrustin and PtALF are two acute-phase proteins involved in the immune responses of P. trituberculatus. PMID:20167286

  11. The Anorexigenic Peptide Neuromedin U (NMU) Attenuates Amphetamine-Induced Locomotor Stimulation, Accumbal Dopamine Release and Expression of Conditioned Place Preference in Mice

    PubMed Central

    Vallöf, Daniel; Vestlund, Jesper; Engel, Jörgen A.; Jerlhag, Elisabet

    2016-01-01

    Amphetamine dependence, besides its substantial economical consequence, is a serious cause of mortality and morbidity. By investigations of the neurochemical correlates through which addictive drugs, such as amphetamine, activate the mesoaccumbal dopamine system unique targets for treatment of drug addiction can be identified. This reward link consists of a dopamine projection from the ventral tegmental area to the nucleus accumbens (NAc) suggesting that these brain areas are important for reward. The physiological function of gut-brain peptides has expanded beyond food intake modulation and involves regulation of drug reinforcement. A novel candidate for reward regulation is the anorexigenic peptide neuromedin U (NMU). We therefore investigated the effects of intracerebroventricular (icv) administration of NMU on amphetamine’s well-documented effects on the mesoaccumbal dopamine system, i.e. locomotor stimulation and accumbal dopamine release in mice. In addition, the effect of accumbal NMU administration on locomotor activity was examined. The effect of NMU, icv or intra-NAc, on the expression of conditioned place preference (CPP) was elucidated. Firstly, we showed that icv administration of NMU attenuate the amphetamine-induced locomotor stimulation, accumbal dopamine release and expression of CPP in mice. Secondly, we found that a lower dose of NMU (icv) reduce the amphetamine-induced locomotor stimulation in mice. Thirdly, we demonstrated that NMU administration into the NAc block the ability of amphetamine to cause a locomotor stimulation in mice. However, accumbal NMU administration did not attenuate the amphetamine-induced expression of CPP in mice. Our novel data suggest that central NMU signalling is involved in development of amphetamine dependence. PMID:27139195

  12. Alpha 7-type nicotinic acetylcholine receptor and prodynorphin mRNA expression after administration of (-)-nicotine and U-50,488H in beta-amyloid peptide (25-35)-treated mice.

    PubMed

    Hiramatsu, M; Watanabe, M; Baba, S; Kojima, R; Nabeshima, T

    2004-10-01

    We previously reported that (-)-nicotine and kappa-opioid receptor agonists lessened impairment of learning and/or memory in several animal models. Furthermore, these drugs prevented neurodegenerative damage induced by ischemia or beta-amyloid peptide (25-35). In the present study, we tested whether (-)-nicotine and U-50,488H prevent delayed-memory impairment induced by beta-amyloid peptide (25-35), and changes of expression of alpha7-type nicotinic acetylcholine receptor mRNA and prodynorphin mRNA. Seven days after treatment with beta-amyloid peptide (25-35) (9 nmol/mouse, i.c.v.), memory impairment was observed in the Y-maze test. Memory impairment was prevented when (-)-nicotine (6.16 micromol/kg, s.c.) or U-50,488H (21 micromol/kg, s.c.) was administered 1 h before, but not 1 h after, beta-amyloid peptide (25-35) treatment. There was no change in prodynorphin mRNA or alpha7-type nicotinic acetylcholine receptor mRNA expression in the hippocampus 10 days after beta-amyloid peptide (25-35) treatment alone. Of interest, mRNA expression of not only prodynorphin, but also the alpha7-type nicotinic acetylcholine receptor, was significantly decreased when U-50,488H was administered 1 h before, but not 1 h after, treatment with beta-amyloid peptide (25-35). However, these changes were not observed after the administration of (-)-nicotine. These results suggest that activation of the kappa-opioid system, but not beta7-type nicotinic receptors has a neuroprotective effect on beta-amyloid peptide (25-35)-induced memory impairment, and may be involved in the long-lasting changes in the expression of these mRNAs.

  13. Over-Expressed Pathogenic miRNAs in Alzheimer's Disease (AD) and Prion Disease (PrD) Drive Deficits in TREM2-Mediated Aβ42 Peptide Clearance.

    PubMed

    Zhao, Yuhai; Jaber, Vivian; Lukiw, Walter J

    2016-01-01

    One prominent and distinguishing feature of progressive, age-related neurological diseases such as Alzheimer's disease (AD) and prion disease (PrD) is the gradual accumulation of amyloids into dense, insoluble end-stage protein aggregates. These polymorphic proteolipid lesions are known to contribute to immunogenic and inflammatory pathology in these insidious and fatal disorders of the human central nervous system (CNS). For example, the evolution of self-aggregating amyloid-beta (Aβ) peptides, such as the 42 amino acid Aβ42 peptide monomer into higher order aggregates are largely due to: (1) the inability of natural processes to clear them from the cellular environment; and/or (2) the overproduction of these amyloid monomers which rapidly mature into higher order oligomers, fibrils and insoluble, end-stage senile plaques. Cells of the CNS such as microglial (MG) cells have evolved essential homeostatic mechanisms to clear Aβ peptides to avoid their accumulation, however, when defective, these clearance mechanisms become overwhelmed and excessive deposition and aggregation of these amyloids result. This 'Perspectives' paper will highlight some emerging concepts on the up-regulation of an inducible microRNA-34a in AD and PrD that drives the down-regulation of the amyloid sensing- and clearance receptor protein TREM2 (the triggering receptor expressed in myeloid/microglial cells). The impairment of this inducible, miRNA-34a-regulated TREM2- and MG-cell based amyloid clearance mechanism may thereby contribute to the age-related amyloidogenesis associated with both AD and PrD. PMID:27378912

  14. Effects of Food-Derived Collagen Peptides on the Expression of Keratin and Keratin-Associated Protein Genes in the Mouse Skin.

    PubMed

    Le Vu, Phuong; Takatori, Ryo; Iwamoto, Taku; Akagi, Yutaka; Satsu, Hideo; Totsuka, Mamoru; Chida, Kazuhiro; Sato, Kenji; Shimizu, Makoto

    2015-01-01

    Oral ingestion of collagen peptides (CP) has long been suggested to exert beneficial effects on the skin, but the molecular events induced by CP on the skin remain unclear. Here, we investigated the effects of oral CP administration on gene expression in hairless mouse skin and of prolyl-hydroxyproline (Pro-Hyp), a collagen-derived dipeptide, on gene expression in a coculture of mouse skin keratinocytes and fibroblasts. Using microarray analysis, we found that oral administration of CP to hairless mice for 6 weeks induced increased expression of Krtap and Krt genes in the skin. Annotation analysis using DAVID revealed that a group of the up-regulated genes, Gprc5d, Sprr2a1, Krt27 and Krtap16-7, is associated with the development of the epidermis and the hair cycle. In addition, the presence of Pro-Hyp (200 μM) induced an increase in the expression of Krtap16-7, Krtap15, Krtap14 and Krtap8-2 in keratinocytes in coculture, partially resembling the in vivo result. The Pro-Hyp-induced up-regulation of these genes was not observed when keratinocytes were cultured without fibroblasts, suggesting that the presence of fibroblasts is essential for the effects of Pro-Hyp. Our study presents new insights into the effects of CP on the skin, which might link to the hair cycle. PMID:25721900

  15. Antimicrobial Peptides Expressed in Medicinal Maggots of the Blow Fly Lucilia sericata Show Combinatorial Activity against Bacteria

    PubMed Central

    Pöppel, Anne-Kathrin; Vogel, Heiko; Wiesner, Jochen

    2015-01-01

    The larvae of the common green bottle fly (Lucilia sericata) produce antibacterial secretions that have a therapeutic effect on chronic and nonhealing wounds. Recent developments in insect biotechnology have made it possible to use these larvae as a source of novel anti-infectives. Here, we report the application of next-generation RNA sequencing (RNA-Seq) to characterize the transcriptomes of the larval glands, crop, and gut, which contribute to the synthesis of antimicrobial peptides (AMPs) and proteins secreted into wounds. Our data confirm that L. sericata larvae have adapted in order to colonize microbially contaminated habitats, such as carrion and necrotic wounds, and are protected against infection by a diverse spectrum of AMPs. L. sericata AMPs include not only lucifensin and lucimycin but also novel attacins, cecropins, diptericins, proline-rich peptides, and sarcotoxins. We identified 47 genes encoding putative AMPs and produced 23 as synthetic analogs, among which some displayed activities against a broad spectrum of microbial pathogens, including Pseudomonas aeruginosa, Proteus vulgaris, and Enterococcus faecalis. Against Escherichia coli (Gram negative) and Micrococcus luteus (Gram positive), we found mostly additive effects but also synergistic activity when selected AMPs were tested in combination. The AMPs that are easy to synthesize are currently being produced in bulk to allow their evaluation as novel anti-infectives that can be formulated in hydrogels to produce therapeutic wound dressings and adhesive bandages. PMID:25666157

  16. Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria.

    PubMed

    Pöppel, Anne-Kathrin; Vogel, Heiko; Wiesner, Jochen; Vilcinskas, Andreas

    2015-05-01

    The larvae of the common green bottle fly (Lucilia sericata) produce antibacterial secretions that have a therapeutic effect on chronic and nonhealing wounds. Recent developments in insect biotechnology have made it possible to use these larvae as a source of novel anti-infectives. Here, we report the application of next-generation RNA sequencing (RNA-Seq) to characterize the transcriptomes of the larval glands, crop, and gut, which contribute to the synthesis of antimicrobial peptides (AMPs) and proteins secreted into wounds. Our data confirm that L. sericata larvae have adapted in order to colonize microbially contaminated habitats, such as carrion and necrotic wounds, and are protected against infection by a diverse spectrum of AMPs. L. sericata AMPs include not only lucifensin and lucimycin but also novel attacins, cecropins, diptericins, proline-rich peptides, and sarcotoxins. We identified 47 genes encoding putative AMPs and produced 23 as synthetic analogs, among which some displayed activities against a broad spectrum of microbial pathogens, including Pseudomonas aeruginosa, Proteus vulgaris, and Enterococcus faecalis. Against Escherichia coli (Gram negative) and Micrococcus luteus (Gram positive), we found mostly additive effects but also synergistic activity when selected AMPs were tested in combination. The AMPs that are easy to synthesize are currently being produced in bulk to allow their evaluation as novel anti-infectives that can be formulated in hydrogels to produce therapeutic wound dressings and adhesive bandages.

  17. Differential cellular expression of organic anion transporting peptides OATP1A2 and OATP2B1 in the human retina and brain: implications for carrier-mediated transport of neuropeptides and neurosteriods in the CNS.

    PubMed

    Gao, Bo; Vavricka, Stephan R; Meier, Peter J; Stieger, Bruno

    2015-07-01

    Organic anion transporting polypeptides (OATPs) are polyspecific organic anion transporters, which are expressed in the blood-brain barrier, the choroid plexus, and other organs. The physiologic function of OATPs in extrahepatic tissues remains ambiguous. In rat retina, members of the OATP family are expressed. We therefore investigated the human retina for the expression of OATP1A2 and OATP2B1 and extended the study to human brain. Furthermore, we searched for peptide neurotransmitters as novel OATP substrates. OATP1A2 displayed a broad expression pattern in human retina as assessed by immunofluorescence localization. It is expressed in photoreceptor bodies and somas of amacrine cells. OATP1B2 expression is restricted to the inner nuclear layer and to the inner plexiform layer. Using paraffin sections from human cortex, cerebellum, and hippocampus, OATP1A2 was localized to neurons and neuronal processes, while OATP2B1 is expressed in endothelial cells of brain capillaries. Substance P and vasoactive intestinal peptide were identified as substrates for OATP1A2 and OATP2B1. Double-labeling immunofluorescence of human retina demonstrated the presence of substance P and of vasoactive intestinal peptides in neurons expressing OATP1A2 and OATP2B1, respectively. The expression of OATP1A2 and OATP2B1 in retinal neurons implies a role of these transporters in the reuptake of peptide neurotransmitters released from retinal neurons. The abundant expression of OATP1A2 in brain neurons points to the possibility that OATP1A2 could be involved in the homeostasis of neurosteroids. The high expression of OATP2B1 in brain capillaries supports an important function of OATPs in substance penetration across the blood-brain barrier.

  18. Macrobrachium rosenbergii mannose binding lectin: synthesis of MrMBL-N20 and MrMBL-C16 peptides and their antimicrobial characterization, bioinformatics and relative gene expression analysis.

    PubMed

    Arockiaraj, Jesu; Chaurasia, Mukesh Kumar; Kumaresan, Venkatesh; Palanisamy, Rajesh; Harikrishnan, Ramasamy; Pasupuleti, Mukesh; Kasi, Marimuthu

    2015-04-01

    Mannose-binding lectin (MBL), an antimicrobial protein, is an important component of innate immune system which recognizes repetitive sugar groups on the surface of bacteria and viruses leading to activation of the complement system. In this study, we reported a complete molecular characterization of cDNA encoded for MBL from freshwater prawn Macrobrachium rosenbergii (Mr). Two short peptides (MrMBL-N20: (20)AWNTYDYMKREHSLVKPYQG(39) and MrMBL-C16: (307)GGLFYVKHKEQQRKRF(322)) were synthesized from the MrMBL polypeptide. The purity of the MrMBL-N20 (89%) and MrMBL-C16 (93%) peptides were confirmed by MS analysis (MALDI-ToF). The purified peptides were used for further antimicrobial characterization including minimum inhibitory concentration (MIC) assay, kinetics of bactericidal efficiency and analysis of hemolytic capacity. The peptides exhibited antimicrobial activity towards all the Gram-negative bacteria taken for analysis, whereas they showed the activity towards only a few selected Gram-positive bacteria. MrMBL-C16 peptides produced the highest inhibition towards both the Gram-negative and Gram-positive bacteria compared to the MrMBL-N20. Both peptides do not produce any inhibition against Bacillus sps. The kinetics of bactericidal efficiency showed that the peptides drastically reduced the number of surviving bacterial colonies after 24 h incubation. The results of hemolytic activity showed that both peptides produced strong activity at higher concentration. However, MrMBL-C16 peptide produced the highest activity compared to the MrMBL-N20 peptide. Overall, the results indicated that the peptides can be used as bactericidal agents. The MrMBL protein sequence was characterized using various bioinformatics tools including phylogenetic analysis and structure prediction. We also reported the MrMBL gene expression pattern upon viral and bacterial infection in M. rosenbergii gills. It could be concluded that the prawn MBL may be one of the important molecule which

  19. Macrobrachium rosenbergii mannose binding lectin: synthesis of MrMBL-N20 and MrMBL-C16 peptides and their antimicrobial characterization, bioinformatics and relative gene expression analysis.

    PubMed

    Arockiaraj, Jesu; Chaurasia, Mukesh Kumar; Kumaresan, Venkatesh; Palanisamy, Rajesh; Harikrishnan, Ramasamy; Pasupuleti, Mukesh; Kasi, Marimuthu

    2015-04-01

    Mannose-binding lectin (MBL), an antimicrobial protein, is an important component of innate immune system which recognizes repetitive sugar groups on the surface of bacteria and viruses leading to activation of the complement system. In this study, we reported a complete molecular characterization of cDNA encoded for MBL from freshwater prawn Macrobrachium rosenbergii (Mr). Two short peptides (MrMBL-N20: (20)AWNTYDYMKREHSLVKPYQG(39) and MrMBL-C16: (307)GGLFYVKHKEQQRKRF(322)) were synthesized from the MrMBL polypeptide. The purity of the MrMBL-N20 (89%) and MrMBL-C16 (93%) peptides were confirmed by MS analysis (MALDI-ToF). The purified peptides were used for further antimicrobial characterization including minimum inhibitory concentration (MIC) assay, kinetics of bactericidal efficiency and analysis of hemolytic capacity. The peptides exhibited antimicrobial activity towards all the Gram-negative bacteria taken for analysis, whereas they showed the activity towards only a few selected Gram-positive bacteria. MrMBL-C16 peptides produced the highest inhibition towards both the Gram-negative and Gram-positive bacteria compared to the MrMBL-N20. Both peptides do not produce any inhibition against Bacillus sps. The kinetics of bactericidal efficiency showed that the peptides drastically reduced the number of surviving bacterial colonies after 24 h incubation. The results of hemolytic activity showed that both peptides produced strong activity at higher concentration. However, MrMBL-C16 peptide produced the highest activity compared to the MrMBL-N20 peptide. Overall, the results indicated that the peptides can be used as bactericidal agents. The MrMBL protein sequence was characterized using various bioinformatics tools including phylogenetic analysis and structure prediction. We also reported the MrMBL gene expression pattern upon viral and bacterial infection in M. rosenbergii gills. It could be concluded that the prawn MBL may be one of the important molecule which

  20. Effects of immobilizations stress with or without water immersion on the expression of atrial natriuretic peptide in the hearts of two rat strains.

    PubMed

    Slavikova, Jana; Mistrova, Eliska; Klenerova, Vera; Kruzliak, Peter; Caprnda, Martin; Hynie, Sixtus; Sida, Pavel; Dvorakova, Magdalena Chottova

    2016-01-01

    Atrial natriuretic peptide (ANP) is produced and released by mammalian cardiomyocytes and induces natriuresis, diuresis, and lowering of blood pressure. The present study examined localization of ANP and a possible role of the hypothalamic-pituitary-adrenal axis (HPA) activity on the expression of proANP gene in the heart. The Sprague Dawley (SD) and Lewis (LE) rat strains were used. The animals were exposed to the two types of stress: immobilization and immobilization combined with water immersion for 1 hour. Localization of ANP was detected by immunohistochemistry and expression of the proANP mRNA by real-time qPCR in all heart compartments of control and stressed animals after 1 and 3 hours after stress termination (IS1, IS3, ICS1, and ICS3). Relatively high density of ANP-immunoreactivity was observed in both atria of both rat strains. In control rats of both strains, the expression of the proANP mRNA was higher in the atria than in ventricles. In SD rats with the intact HPA axis, an upregulation of ANP gene expression was observed in the right atrium after IS1, in both atria and the left ventricle after IS3 and in the left atrium and the left ventricle after ICS3. In LE rats with a blunted reactivity of the HPA axis, no increase or even a downregulation of the gene expression was observed. Thus, acute stress-induced increase in the expression of the proANP gene is related to the activity of the HPA axis. It may have relevance to ANP-induced protection of the heart.

  1. Effects of immobilizations stress with or without water immersion on the expression of atrial natriuretic peptide in the hearts of two rat strains

    PubMed Central

    Slavikova, Jana; Mistrova, Eliska; Klenerova, Vera; Kruzliak, Peter; Caprnda, Martin; Hynie, Sixtus; Sida, Pavel; Dvorakova, Magdalena Chottova

    2016-01-01

    Atrial natriuretic peptide (ANP) is produced and released by mammalian cardiomyocytes and induces natriuresis, diuresis, and lowering of blood pressure. The present study examined localization of ANP and a possible role of the hypothalamic-pituitary-adrenal axis (HPA) activity on the expression of proANP gene in the heart. The Sprague Dawley (SD) and Lewis (LE) rat strains were used. The animals were exposed to the two types of stress: immobilization and immobilization combined with water immersion for 1 hour. Localization of ANP was detected by immunohistochemistry and expression of the proANP mRNA by real-time qPCR in all heart compartments of control and stressed animals after 1 and 3 hours after stress termination (IS1, IS3, ICS1, and ICS3). Relatively high density of ANP-immunoreactivity was observed in both atria of both rat strains. In control rats of both strains, the expression of the proANP mRNA was higher in the atria than in ventricles. In SD rats with the intact HPA axis, an upregulation of ANP gene expression was observed in the right atrium after IS1, in both atria and the left ventricle after IS3 and in the left atrium and the left ventricle after ICS3. In LE rats with a blunted reactivity of the HPA axis, no increase or even a downregulation of the gene expression was observed. Thus, acute stress-induced increase in the expression of the proANP gene is related to the activity of the HPA axis. It may have relevance to ANP-induced protection of the heart. PMID:27508036

  2. Effects of immobilizations stress with or without water immersion on the expression of atrial natriuretic peptide in the hearts of two rat strains.

    PubMed

    Slavikova, Jana; Mistrova, Eliska; Klenerova, Vera; Kruzliak, Peter; Caprnda, Martin; Hynie, Sixtus; Sida, Pavel; Dvorakova, Magdalena Chottova

    2016-01-01

    Atrial natriuretic peptide (ANP) is produced and released by mammalian cardiomyocytes and induces natriuresis, diuresis, and lowering of blood pressure. The present study examined localization of ANP and a possible role of the hypothalamic-pituitary-adrenal axis (HPA) activity on the expression of proANP gene in the heart. The Sprague Dawley (SD) and Lewis (LE) rat strains were used. The animals were exposed to the two types of stress: immobilization and immobilization combined with water immersion for 1 hour. Localization of ANP was detected by immunohistochemistry and expression of the proANP mRNA by real-time qPCR in all heart compartments of control and stressed animals after 1 and 3 hours after stress termination (IS1, IS3, ICS1, and ICS3). Relatively high density of ANP-immunoreactivity was observed in both atria of both rat strains. In control rats of both strains, the expression of the proANP mRNA was higher in the atria than in ventricles. In SD rats with the intact HPA axis, an upregulation of ANP gene expression was observed in the right atrium after IS1, in both atria and the left ventricle after IS3 and in the left atrium and the left ventricle after ICS3. In LE rats with a blunted reactivity of the HPA axis, no increase or even a downregulation of the gene expression was observed. Thus, acute stress-induced increase in the expression of the proANP gene is related to the activity of the HPA axis. It may have relevance to ANP-induced protection of the heart. PMID:27508036

  3. Expression of trefoil peptides (TFF1, TFF2, and TFF3) in gastric carcinomas, intestinal metaplasia, and non-neoplastic gastric tissues.

    PubMed

    Leung, Wai K; Yu, Jun; Chan, Francis K L; To, Ka F; Chan, Michael W Y; Ebert, M P A; Ng, Enders K W; Chung, S C Sydney; Malfertheiner, Peter; Sung, Joseph J Y

    2002-08-01

    Trefoil factor family (TFF) domain peptides consist of three members that play a role in intestinal mucosal defence and repair, and in tumourigenesis. The role of the three TFF members in the gastric carcinogenesis cascade remains poorly defined. This study examined seven gastric cell lines, 50 gastric cancers and their adjacent non-cancer tissues, and tissues from 40 non-cancer patients, in order to elucidate the chronology of TFF expression in various stages of gastric carcinogenesis. TFF expression was determined by RT-PCR, immunohistochemistry, and western blot. Aberrant expression of TFF1, TFF2, and TFF3 was frequently detected in gastric cell lines. Specifically, TFF1 was detected in all non-cancer patients, but was detected in only 50% of gastric cancer and 66% of adjacent normal tissues. TFF2 expression was demonstrated in 87.5% of non-cancer patients, 34% of gastric carcinomas, and 58% of adjacent non-cancer tissues. There was a significant correlation between TFF1 and TFF2 expression in gastric cancer and adjacent non-cancer tissues (p<0.001). By contrast, TFF3 was detected in 25% of non-cancer patients and showed a predilection for areas with intestinal metaplasia (p=0.005). Sixty-two per cent of gastric cancers and 24% of neighbouring non-cancer tissues showed TFF3 expression. Immunoreactivity against TFF3 was demonstrated in goblet cells of intestinal metaplasia and within the cytoplasm and nuclei of tumour cells. Progressive loss of TFF1 and TFF2, together with the induction of TFF3, is likely to be involved in the early stage of the multi-step gastric carcinogenesis pathway.

  4. PcToll3 was involved in anti-Vibrio response by regulating the expression of antimicrobial peptides in red swamp crayfish, Procambarus clarkii.

    PubMed

    Lan, Jiang-Feng; Wei, Shun; Wang, Yu-Qing; Dai, Yun-Jia; Tu, Jia-Gang; Zhao, Li-Juan; Li, Xin-Cang; Qin, Qi-Wei; Chen, Nan; Lin, Li

    2016-10-01

    Tolls and Toll-like receptors (TLRs) play an important role in host immune defenses by regulating the expression of antimicrobial peptides (AMPs) and cytokines, but the functional differences of crustacean Tolls from Drosophila Tolls or Mammal TLRs are largely unknown. A novel Toll receptor, named PcToll3, was identified from red swamp crayfish, Procambarus clarkii. It was widely expressed in all detected tissues, and its transcript in hemocytes was up-regulated at 12 h after Vibrio parahemolyticus (Vibrio) injection or at 24 h post white spot syndrome virus (WSSV) challenge. After knockdown of PcToll3, the activity of bacterial clearance was inhibited, and the expression levels of AMPs including Crustin1 (Cru1), Anti-lippopolysaccharide factor 1 (ALF1), and Lysozymes1 (Lys1), which could be up-regulated by Vibrio, were all affected. Meanwhile, PcToll3 silencing influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of Drosophila Toll signaling pathway. Interestingly, PcToll3 silencing inhibited translocation of PcDorsal from cytoplasm to nucleus. Furthermore, the knockdown of PcDorsal also impaired the expression of AMPs after Vibrio challenge. Hence, we concluded that, besides participating in antiviral immunity, PcToll3 might also regulate the expression of Cru1 and Lys1 to participate in anti-Vibrio immune responses by promoting PcDorsal translocation into nucleus. PMID:27531577

  5. PcToll3 was involved in anti-Vibrio response by regulating the expression of antimicrobial peptides in red swamp crayfish, Procambarus clarkii.

    PubMed

    Lan, Jiang-Feng; Wei, Shun; Wang, Yu-Qing; Dai, Yun-Jia; Tu, Jia-Gang; Zhao, Li-Juan; Li, Xin-Cang; Qin, Qi-Wei; Chen, Nan; Lin, Li

    2016-10-01

    Tolls and Toll-like receptors (TLRs) play an important role in host immune defenses by regulating the expression of antimicrobial peptides (AMPs) and cytokines, but the functional differences of crustacean Tolls from Drosophila Tolls or Mammal TLRs are largely unknown. A novel Toll receptor, named PcToll3, was identified from red swamp crayfish, Procambarus clarkii. It was widely expressed in all detected tissues, and its transcript in hemocytes was up-regulated at 12 h after Vibrio parahemolyticus (Vibrio) injection or at 24 h post white spot syndrome virus (WSSV) challenge. After knockdown of PcToll3, the activity of bacterial clearance was inhibited, and the expression levels of AMPs including Crustin1 (Cru1), Anti-lippopolysaccharide factor 1 (ALF1), and Lysozymes1 (Lys1), which could be up-regulated by Vibrio, were all affected. Meanwhile, PcToll3 silencing influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of Drosophila Toll signaling pathway. Interestingly, PcToll3 silencing inhibited translocation of PcDorsal from cytoplasm to nucleus. Furthermore, the knockdown of PcDorsal also impaired the expression of AMPs after Vibrio challenge. Hence, we concluded that, besides participating in antiviral immunity, PcToll3 might also regulate the expression of Cru1 and Lys1 to participate in anti-Vibrio immune responses by promoting PcDorsal translocation into nucleus.

  6. Leptin signaling in GFAP-expressing adult glia cells regulates hypothalamic neuronal circuits and feeding

    PubMed Central

    Kim1, Jae Geun; Suyama, Shigetomo; Koch, Marco; Jin, Sungho; Argente-Arizon, Pilar; Argente, Jesus; Liu, Zhong-Wu; Zimmer, Marcelo R.; Jeong, Jin Kwon; Szigeti-Buck, Klara; Gao, Yuanqing; Garcia-Caceres, Cristina; Yi, Chun-Xia; Salmaso, Natalina; Vaccarino, Flora M.; Chowen, Julie; Diano, Sabrina; Dietrich, Marcelo O; Tschöp, Matthias H.; Horvath, Tamas L.

    2014-01-01

    We have shown that synaptic re-organization of hypothalamic feeding circuits in response to metabolic shifts involves astrocytes, cells that can directly respond to the metabolic hormone, leptin, in vitro. It is not known whether the role of glia cells in hypothalamic synaptic adaptions is active or passive. Here we show that leptin receptors are expressed in hypothalamic astrocytes and that conditional, adult deletion of leptin receptors in astrocytes leads to altered glial morphology, decreased glial coverage and elevated synaptic inputs onto pro-opiomelanocortin (POMC)- and Agouti-related protein (AgRP)-producing neurons. Leptin-induced suppression of feeding was diminished, while rebound feeding after fasting or ghrelin administration was elevated in mice with astrocyte-specific leptin receptor deficiency. These data unmask an active role of glial cells in the initiation of hypothalamic synaptic plasticity and neuroendocrine control of feeding by leptin. PMID:24880214

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

    PubMed Central

    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

  8. IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides

    PubMed Central

    2011-01-01

    The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity. PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity. IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously. These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression

  9. Expression profiling of a diapause-specific peptide (DSP) of the leaf beetle Gastrophysa atrocyanea and silencing of DSP by double-strand RNA.

    PubMed

    Tanaka, Hiromasa; Suzuki, Koichi

    2005-06-01

    A diapause-specific peptide (DSP) composed of 41 amino acid residues including 6 cysteines, has been isolated from diapausing adults of the leaf beetle Gastrophysa atrocyanea. In this study, DSP was found to be expressed primarily in diapausing adults and to a minor extent in pupae, but not in eggs, larvae, or post-diapausing adults. DSP was not induced by bacterial or fungal challenge. DSP-less adults were generated by the injection of double-stranded RNA (dsRNA) corresponding to the dsp gene into pre-diapausing adults. Gene silencing induced by dsRNA was found to be a useful tool for the analysis of DSP in diapausing adults. DSP-less adults showed similar burrowing behavior and oxygen consumption as control insects suggesting that DSP is not essential for the normal onset and maintenance of diapause.

  10. Supplementation with branched-chain amino acids to a low-protein diet regulates intestinal expression of amino acid and peptide transporters in weanling pigs.

    PubMed

    Zhang, Shihai; Qiao, Shiyan; Ren, Man; Zeng, Xiangfang; Ma, Xi; Wu, Zhenlong; Thacker, Philip; Wu, Guoyao

    2013-11-01

    This study determined the effects of dietary branched-chain amino acids (AA) (BCAA) on growth performance, expression of jejunal AA and peptide transporters, and the colonic microflora of weanling piglets fed a low-protein (LP) diet. One hundred and eight Large White × Landrace × Duroc piglets (weaned at 28 days of age) were fed a normal protein diet (NP, 20.9 % crude protein), an LP diet (LP, 17.1 % crude protein), or an LP diet supplemented with BCAA (LP + BCAA, 17.9 % crude protein) for 14 days. Dietary protein restriction reduced piglet growth performance and small-intestinal villous height, which were restored by BCAA supplementation to the LP diet to values for the NP diet. Serum concentrations of BCAA were reduced in piglets fed the LP diet while those in piglets fed the LP + BCAA diet were similar to values for the NP group. mRNA levels for Na(+)-neutral AA exchanger-2, cationic AA transporter-1, b(0,+) AA transporter, and 4F2 heavy chain were more abundant in piglets fed the LP + BCAA diet than the LP diet. However, mRNA and protein levels for peptide transporter-1 were lower in piglets fed the LP + BCAA diet as compared to the LP diet. The colonic microflora did not differ among the three groups of pigs. In conclusion, growth performance, intestinal development, and intestinal expression of AA transporters in weanling piglets are enhanced by BCAA supplementation to LP diets. Our findings provide a new molecular basis for further understanding of BCAA as functional AA in animal nutrition.

  11. Vitamin D3 Suppresses Class II Invariant Chain Peptide Expression on Activated B-Lymphocytes: A Plausible Mechanism for Downregulation of Acute Inflammatory Conditions.

    PubMed

    Danner, Omar K; Matthews, Leslie R; Francis, Sharon; Rao, Veena N; Harvey, Cassie P; Tobin, Richard P; Wilson, Ken L; Alema-Mensah, Ernest; Newell Rogers, M Karen; Childs, Ed W

    2016-01-01

    Class II invariant chain peptide (CLIP) expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP(+) B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI) based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol) had the most dramatic reduction in CLIP density at an MFI of 257.3 [baseline of 701.1 (P value = 0.01)]. Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings.

  12. β-Arrestin 1’s Interaction with TC45 Attenuates Stat signaling by dephosphorylating Stat to inhibit antimicrobial peptide expression

    PubMed Central

    Sun, Jie-Jie; Yang, Hui-Ting; Niu, Guo-Juan; Feng, Xiao-Wu; Lan, Jiang-Feng; Zhao, Xiao-Fan; Wang, Jin-Xing

    2016-01-01

    Impaired phosphatase activity leads to the persistent activation of signal transducers and activators of transcription (Stat). In mammals, Stat family members are often phosphorylated or dephosphorylated by the same enzymes. To date, only one Stat similar to mammalian Stat5a/b has been found in crustaceans and there have been few studies in Stat signal regulation in crustaceans. Here, we report that β-arrestin1 interacts with TC45 (45-kDa form of T cell protein tyrosine phosphatase) in the nucleus to attenuate Stat signaling by promoting dephosphorylation of Stat. Initially, we showed that Stat translocates into the nucleus to induce antimicrobial peptide (AMP) expression after bacterial infection. βArr1 enters the nucleus of hemocytes and recruits TC45 to form the βarr1-TC45-Stat complex, which dephosphorylates Stat efficiently. The interaction of TC45 with Stat decreased and Stat phosphorylation increased in βarr1-silenced shrimp (Marsupenaeus japonicus) after challenge with Vibrio anguillarum. βArr1 directly interacts with Stat in nucleus and accelerates Stat dephosphorylation by recruiting TC45 after V. anguillarum challenge. Further study showed that βarr1 and TC45 also affect AMP expression, which is regulated by Stat. Therefore, βarr1 and TC45 are involved in the anti-V. anguillarum immune response by regulating Stat activity negatively to decrease AMP expression in shrimp. PMID:27782165

  13. Oral delivery of probiotic expressing M cell homing peptide conjugated BmpB vaccine encapsulated into alginate/chitosan/alginate microcapsules.

    PubMed

    Jiang, Tao; Singh, Bijay; Maharjan, Sushila; Li, Hui-Shan; Kang, Sang-Kee; Bok, Jin-Duck; Cho, Chong-Su; Choi, Yun-Jaie

    2014-11-01

    Oral administration of live probiotics as antigen delivery vectors is a promising approach in vaccine development. However, the low survival of probiotics in the gastrointestinal tract limits this approach. Therefore, the aim of this study was the encapsulation of probiotic expressing vaccine into alginate/chitosan/alginate (ACA) microcapsules (MCs) for efficient oral vaccine delivery. Here, recombinant Lactobacillus plantarum 25 (LP25) expressing M cell homing peptide fused BmpB protein was used as a model probiotic. The viability of LP25 in ACA MCs was more than 65% in simulated gastric fluid (SGF, pH 2.0) and 75% in simulated small intestinal fluid (SIF, pH 7.2) up to 2h. Encapsulated LP25 was completely released from ACA MCs in SIF within 12h. When stored at room temperature (RT) or 4°C, the viability of LP25 in ACA MCs was higher than free LP25. Interestingly, the viability of LP25 in ACA MCs at 4°C for 5weeks was above 58%, whereas viability of free LP25 stored at RT up to 5weeks was zero. After 4weeks from the first immunization, LP25-M-BmpB-loaded ACA MCs induced a stronger BmpB-specific IgG and IgA production in mice. Collectively, these findings suggest that encapsulation of probiotic by ACA MCs is a promising delivery system for oral administration of probiotic expressing vaccine.

  14. Vitamin D3 Suppresses Class II Invariant Chain Peptide Expression on Activated B-Lymphocytes: A Plausible Mechanism for Downregulation of Acute Inflammatory Conditions.

    PubMed

    Danner, Omar K; Matthews, Leslie R; Francis, Sharon; Rao, Veena N; Harvey, Cassie P; Tobin, Richard P; Wilson, Ken L; Alema-Mensah, Ernest; Newell Rogers, M Karen; Childs, Ed W

    2016-01-01

    Class II invariant chain peptide (CLIP) expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP(+) B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI) based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol) had the most dramatic reduction in CLIP density at an MFI of 257.3 [baseline of 701.1 (P value = 0.01)]. Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings. PMID:27313879

  15. Vitamin D3 Suppresses Class II Invariant Chain Peptide Expression on Activated B-Lymphocytes: A Plausible Mechanism for Downregulation of Acute Inflammatory Conditions

    PubMed Central

    Danner, Omar K.; Matthews, Leslie R.; Francis, Sharon; Rao, Veena N.; Harvey, Cassie P.; Tobin, Richard P.; Wilson, Ken L.; Alema-Mensah, Ernest; Newell Rogers, M. Karen; Childs, Ed W.

    2016-01-01

    Class II invariant chain peptide (CLIP) expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP+ B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI) based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol) had the most dramatic reduction in CLIP density at an MFI of 257.3 [baseline of 701.1 (P value = 0.01)]. Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings. PMID:27313879

  16. High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants.

    PubMed

    Firsov, Aleksey; Tarasenko, Irina; Mitiouchkina, Tatiana; Ismailova, Natalya; Shaloiko, Lyubov; Vainstein, Alexander; Dolgov, Sergey

    2015-07-01

    Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.

  17. Activity, Expression and Genetic Variation of Canine β-Defensin 103: A Multifunctional Antimicrobial Peptide in the Skin of Domestic Dogs

    PubMed Central

    Leonard, Brian C.; Marks, Stanley L.; Outerbridge, Catherine A.; Affolter, Verena K.; Kananurak, Anchasa; Young, Amy; Moore, Peter F.; Bannasch, Danika L.; Bevins, Charles L.

    2012-01-01

    The skin functions as more than a physical barrier to infection. Epithelial cells of the skin can synthesize antimicrobial peptides, including defensins, which exhibit direct antimicrobial activity. Here we characterize the expression pattern, genetic variation and activity of the major β-defensin expressed in canine skin, canine β-defensin 103 (CBD103). The gene encoding CBD103 exhibits two forms of polymorphism: a common 3-basepair deletion allele and a gene copy-number variation. Golden retrievers and Labrador retrievers were the only breeds that encoded the variant allele of CBD103, termed CBD103ΔG23. Both these breeds also exhibited a CBD103 gene copy-number polymorphism that ranged from 2 to 4 gene-copies per diploid genome. Recombinant CBD103 and CBD103ΔG23, as well as the human ortholog human β-defensin 3 (hBD3) and hBD3ΔG23, showed potent and comparable antimicrobial killing against both methicillin-susceptible and methicillin-resistant Staphylococcus pseudintermedius. Skin biopsy specimens from dogs with atopic dermatitis revealed CBD103 expression levels similar to those in healthy controls and comparable at lesional and nonlesional sites. This expression pattern in dogs differs from the previously reported reduced expression of the human ortholog in atopic dermatitis. Overall, the similarities of CBD103 and its human ortholog reported here support the notion that the domestic dog may serve as a valuable model for studying β-defensin biology in the skin. PMID:22261569

  18. Bile Acid-regulated Peroxisome Proliferator-activated Receptor-α (PPARα) Activity Underlies Circadian Expression of Intestinal Peptide Absorption Transporter PepT1/Slc15a1*

    PubMed Central

    Okamura, Ayako; Koyanagi, Satoru; Dilxiat, Adila; Kusunose, Naoki; Chen, Jia Jun; Matsunaga, Naoya; Shibata, Shigenobu; Ohdo, Shigehiro

    2014-01-01

    Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter. PMID:25016014

  19. Bile acid-regulated peroxisome proliferator-activated receptor-α (PPARα) activity underlies circadian expression of intestinal peptide absorption transporter PepT1/Slc15a1.

    PubMed

    Okamura, Ayako; Koyanagi, Satoru; Dilxiat, Adila; Kusunose, Naoki; Chen, Jia Jun; Matsunaga, Naoya; Shibata, Shigenobu; Ohdo, Shigehiro

    2014-09-01

    Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter.

  20. The mRNA expression of amino acid transporters, aminopeptidase N, and the di- and tri- peptide transporter PepT1 in the embryo of the domesticated chicken (Gallus gallus) shows developmental regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mRNA expression profile for ten amino acid transporters (AAT), the di-and tri- peptide transporter (Pept1), and aminopeptidase N (APN) during chick embryogenesis was determined. Fertilized eggs were sampled at days 9, 11, 15, 17, 19, and 20, post fertilization. Three to four embryos were sampl...

  1. Heterologous expressed toxic and non-toxic peptide variants of toxin CssII are capable to produce neutralizing antibodies against the venom of the scorpion Centruroides suffusus suffusus.

    PubMed

    Hernández-Salgado, Kenya; Estrada, Georgina; Olvera, Alejandro; Coronas, Fredy I; Possani, Lourival D; Corzo, Gerardo

    2009-08-15

    Two toxic and one non-toxic recombinant peptide variants of the mammalian neurotoxin CssII was cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage site. The toxic recombinant peptides rCssII, HisrCssII and the non-toxic rCssIIE15R were expressed under induction with isopropyl thiogalactoside (IPTG), isolated using chromatographic techniques and folded correctly in vitro. The three recombinant variants showed similar secondary structures as the native CssII, but only the rCssIIE15R was not toxic to mice at concentrations up to 30microg/20g mouse body weight when injected intraperitoneally. All three recombinant peptides were capable of displacing the native CssII from their receptor sites in rat brain synaptosomes, suggesting that they had similar structural and functional characteristics of the native peptides. The three recombinant variants of CssII and the native one were used as antigens for immunization of New Zealand rabbits. The antibodies present in the rabbit antisera were able to recognize the native CssII. Additionally and more importantly, the sera of the immunized rabbits were able to neutralize both the native toxin CssII and the whole soluble venom of the scorpion Centruroides suffusus suffusus. These results indicate that the recombinant peptides can be used to produce antidotes against the venom of this species of scorpion.

  2. Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo.

    PubMed

    Chen, Joy; Peterson, Kenneth R; Iancu-Rubin, Camelia; Bieker, James J

    2010-09-28

    Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNA-binding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.

  3. Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells

    PubMed Central

    ZHOU, RI; YUAN, ZHI; LIU, JIERONG; LIU, JIAN

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/β-catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/β-catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/β-catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/β-catenin pathway, including the mRNA of c-myc, cyclin D1, Lef1, Tcf7 and β-catenin as well as β-catenin protein. However, the upregulation of these genes and β-catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled-related protein, an antagonist of the Wnt/β-catenin pathway. The results of the present study therefore suggested that the Wnt/β-catenin signaling pathway may be involved in CGRP- and LiCl-promoted osteoblastic differentiation of BMSCs. PMID:27082317

  4. Genetically Altered Mutant Mouse Models of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Exhibit the Cardiac Expression of Proinflammatory Mediators in a Gene-Dose-Dependent Manner

    PubMed Central

    Vellaichamy, Elangovan; Das, Subhankar; Subramanian, Umadevi; Maeda, Nobuyo

    2014-01-01

    The objective of this study was to examine whether genetically determined differences in the guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1) affect cardiac expression of proinflammatory cytokines, hypertrophic markers, nuclear factor-κB (NF-κB), and activating protein-1 (AP-1) in am Npr1 gene-dose–dependent manner. In the present studies, adult male Npr1 gene-disrupted (Npr1−/−), wild-type (Npr1+/+), and gene-duplicated (Npr1++/++) mice were used. The Npr1−/− mice showed 41 mm Hg higher systolic blood pressure and 60% greater heart weight to body weight (HW/BW) ratio; however, Npr1++/++ mice exhibited 15 mm Hg lower systolic blood pressure and 12% reduced HW/BW ratio compared with Npr1+/+ mice. Significant upregulation of gene expression of proinflammatory cytokines and hypertrophic markers along with enhanced NF-κB/AP-1 binding activities were observed in the Npr1−/− mouse hearts. Conversely, hypertrophic markers and proinflammatory cytokines gene expression as well as NF-κB/AP-1 binding activities were markedly decreased in Npr1++/++ mouse hearts compared with wild-type mice. The ventricular guanylyl cyclase activity and cGMP levels were reduced by 96% and 87%, respectively, in Npr1−/− mice; however, these parameters were amplified by 2.8-fold and 3.8-fold, respectively, in Npr1++/++ mice. Echocardiographic analysis revealed significantly increased fractional shortening in Npr1++/++ mice (P < .05) but greatly decreased in Npr1−/− mice (P < .01) hearts compared with Npr1+/+ mice. The present findings suggest that Npr1 represses the expression of cardiac proinflammatory mediators, hypertrophic markers, and NF-κB/AP-1–mediated mechanisms, which seem to be associated in an Npr1 gene-dose–dependent manner. PMID:24424043

  5. Preliminary X-ray diffraction analysis of crystals from the recombinantly expressed human major histocompatibility antigen HLA-B*2704 in complex with a viral peptide and with a self-peptide

    SciTech Connect

    Loll, Bernhard; Biesiadka, Jacek; Saenger, Wolfram

    2005-10-01

    Crystallization of HLA-B*2704 in complex with two peptides. The product of the human leukocyte antigen (HLA) gene HLA-B*2704 differs from that of the prototypical subtype HLA-B*2705 by three amino acids at heavy-chain residues 77 (Ser instead of Asp), 152 (Glu instead of Val) and 211 (Gly instead of Ala). In contrast to the ubiquitous HLA-B*2705 subtype, HLA-B*2704 occurs only in orientals. Both subtypes are strongly associated with spondyloarthropathies and the peptides presented by these subtypes are suspected to play a role in disease pathogenesis. HLA-B*2704 was crystallized in complex with a viral peptide and with a self-peptide using the hanging-drop vapour-diffusion method with PEG as a precipitant. Both crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}. Data sets were collected to 1.60 Å (complex with the self-peptide pVIPR) or to 1.90 Å (complex with the viral peptide pLMP2) resolution using synchrotron radiation. With HLA-B*2705 complexed with pVIPR as a search model, unambiguous molecular-replacement solutions were found for the complexes of HLA-B*2704 with both peptides.

  6. Differential expression of thrombospondin (THBS1) in tumorigenic and nontumorigenic prostate epithelial cells in response to a chromatin-binding soy peptide.

    PubMed

    Galvez, Alfredo F; Huang, Liping; Magbanua, Mark M J; Dawson, Kevin; Rodriguez, Raymond L

    2011-01-01

    The chemopreventive properties of the chromatin-binding soy peptide, lunasin, are well documented, but its mechanism of action is unclear. To elucidate the mechanism by which lunasin reduces tumor foci formation in cultured mammalian cells, nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells were treated with lunasin followed by gene expression profiling and characterization of the chromatin acetylation status for certain chemopreventive genes. The genes HIF1A, PRKAR1A, TOB1, and THBS1 were upregulated by lunasin in RWPE-1 but not in RWPE-2 cells. Using histone acetyltransferase (HAT) assays with acid-extracted histones as templates, we showed that lunasin specifically inhibited H4K8 acetylation while enhanced H4K16 acetylation catalyzed by HAT enzymes p300, PCAF, and HAT1A. These results suggest a novel mechanism for lunasin-dependent upregulation of gene expression. Chromatin immunoprecipitation (ChIP) revealed hypoacetylation of H4K16 in RWPE-2 cells, specifically at the 5' end of THBS1 containing a CpG island. Moreover, bisulfite PCR (BSP) and subsequent DNA sequencing indicated that this CpG island was hypomethylated in RWPE-1 but hypermethylated in RWPE-2 cells. Histone hypoacetylation and DNA hypermethylation in the 5' region of THBS1 may explain the inability of lunasin to upregulate this gene in RWPE-2 cells. PMID:21526452

  7. Artificial antigen-presenting cells expressing AFP158-166 peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes

    PubMed Central

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; Zhang, Zhixiang; He, Xianghui

    2016-01-01

    Professional antigen-presenting cells (APCs) are potent generators of tumor antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy; however, generation of APCs is cumbersome, expensive, and subject to the tumor microenvironment. Artificial APCs (aAPCs) have been developed as a cost-effective alternative to APCs. We developed a cellular aAPC that efficiently generated alpha-fetoprotein (AFP)-specific CTLs. We genetically modified the human B cell lymphoma cell line BJAB with a lentiviral vector to establish an aAPC called BA15. The expression of AFP158-166-HLA-A*02:01 complex, CD80, CD86, and interleukin (IL)-15 in BA15 cells was assessed. The efficiency of BA15 at generating AFP-specific CTLs and the specific cytotoxicity of CTLs against AFP+ cells were also determined. BA15 cells expressed high levels of AFP158-166 peptide, HLA-A2, CD80, CD86, and IL-15. BA15 cells also exhibited higher efficiency in generating AFP-specific CTLs than did dendritic cells. These CTLs had greater cytotoxicity against AFP+ hepatocellular carcinoma cells than did CTLs obtained from dendritic cells in vitro and in vivo. Our novel aAPC system could provide a robust platform for the generation of functional AFP-specific CTLs for adoptive immunotherapy of hepatocellular carcinoma. PMID:27007051

  8. A novel C-type lectin with four CRDs is involved in the regulation of antimicrobial peptide gene expression in Hyriopsis cumingii.

    PubMed

    Zhao, Ling-Ling; Wang, Yu-Qing; Dai, Yun-Jia; Zhao, Li-Juan; Qin, Qiwei; Lin, Li; Ren, Qian; Lan, Jiang-Feng

    2016-08-01

    C-type lectins (CTLs) are found in a wide number of invertebrates, and have been reported to participate in immune responses, such as the activation of prophenoloxidase, cell adhesion, bacterial clearance and phagocytosis. Previous studies on CTLs focused on the function of their carbohydrate recognition domains (CRDs). Currently, studies on lectins with multi-CRDs are limited. In this study, a lectin with four CRDs was cloned from Hyriopsis cumingii, and called HcLec4. HcLec4 was widely distributed in several tissues and was significantly down-regulated at the early stage (2 h) of bacterial infection. We further analyzed the bacteria and carbohydrate binding activities of HcLec4. The results showed that HcLec4 could bind to several bacteria, lipopolysaccharide (LPS) and peptidoglycan (PGN). In HcLec4 knockdown mussels, the bacterial clearance rate was increased, and the expression level of antimicrobial peptides (AMPs) was up-regulated. This study reveals that HcLec4 exerts its antibacterial effect by regulating the expression of AMPs at the early stage of bacterial infection. PMID:27288254

  9. Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

    PubMed

    Panadero, Joaquin; Randez-Gil, Francisca; Prieto, Jose Antonio

    2005-12-28

    The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions. PMID:16366681

  10. Chimeras of mature pediocin PA-1 fused to the signal peptide of enterocin P permits the cloning, production, and expression of pediocin PA-1 in Lactococcus lactis.

    PubMed

    Martín, María; Gutiérrez, Jorge; Criado, Raquel; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2007-12-01

    Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SP(entP)) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SP(entP):pedA) and pMPP14i (SP(entP):pedA + pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti-PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SP(EntP) is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.

  11. Effects of dietary supplementation with an expressed fusion peptide bovine lactoferricin-lactoferrampin on performance, immune function and intestinal mucosal morphology in piglets weaned at age 21 d.

    PubMed

    Tang, Zhiru; Yin, Yulong; Zhang, Youming; Huang, Ruilin; Sun, Zhihong; Li, Tiejun; Chu, Wuying; Kong, Xiangfeng; Li, Lili; Geng, Meimei; Tu, Qiang

    2009-04-01

    Lactoferrin has antimicrobial activity associated with peptide fragments lactoferricin (LFC) and lactoferrampin (LFA) released on digestion. These two fragments have been expressed in Photorhabdus luminescens as a fusion peptide linked to protein cipB. The construct cipB-LFC-LFA was tested as an alternative to antimicrobial growth promoters in pig production. Sixty piglets with an average live body weight of 5.42 (sem 0.59) kg were challenged with enterotoxigenic Escherichia coli and randomly assigned to four treatment groups fed a maize-soyabean meal diet containing either no addition (C), cipB at 100 mg/kg (C+B), cipB-LFC-LFA at 100 mg/kg (C+L) or colistin sulfate at 100 mg/kg (C+CS) for 3 weeks. Compared with C, dietary supplementation with C+L for 3 weeks increased daily weight gain by 21 %, increased recovery from diarrhoea, enhanced serum glutathione peroxidase (GPx), peroxidase (POD) and total antioxidant content (T-AOC), liver GPx, POD, superoxide dismutase and T-AOC, Fe, total Fe-binding capacity, IgA, IgG and IgM levels (P < 0.05), decreased the concentration of E. coli in the ileum, caecum and colon (P < 0.05), increased the concentration of lactobacilli and bifidobacteria in the ileum, caecum and colon (P < 0.05), and promoted development of the villus-crypt architecture of the small intestine. Growth performance was similar between C+L- and C+CS-supplemented pigs. The present results indicate that LFC-LFA is an effective alternative to the feed antibiotic CS for enhancing growth performance in piglets weaned at age 21 d. PMID:18840311

  12. Positions of Trp codons in the leader peptide-coding region of the at operon influence anti-trap synthesis and trp operon expression in Bacillus licheniformis.

    PubMed

    Levitin, Anastasia; Yanofsky, Charles

    2010-03-01

    Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNA(Trp). Tryptophan-activated TRAP binds to trp operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNA(Trp). In this paper, we focus on explaining the differences in organization and regulatory functions of the at operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNA(Trp) deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis. PMID:20061467

  13. A polysaccharide-peptide complex from abalone mushroom (Pleurotus abalonus) fruiting bodies increases activities and gene expression of antioxidant enzymes and reduces lipid peroxidation in senescence-accelerated mice.

    PubMed

    Li, L; Ng, T B; Song, M; Yuan, F; Liu, Z K; Wang, C L; Jiang, Y; Fu, M; Liu, F

    2007-06-01

    The antioxidant effects of a polysaccharide-peptide complex (F22) from mushroom (Pleurotus abalonus)-fruiting bodies were studied. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the liver, kidney, and brain of senescence-accelerated mice showed a marked increase after treatment with the polysaccharide-peptide complex. Concurrently, the gene expression levels of SOD, CAT, and GPx, as determined with real-time polymerase chain reaction, were up-regulated in the liver, kidney, and brain, whereas the MDA content in these organs declined. The maximal lifespan of the mice was prolonged.

  14. mTORC1 signaling in Agrp neurons mediates circadian expression of Agrp and NPY but is dispensable for regulation of feeding behavior.

    PubMed

    Albert, Verena; Cornu, Marion; Hall, Michael N

    2015-08-21

    Orexigenic agouti-related protein/neuropeptide Y (Agrp/NPY) neurons and an orexigenic pro-opiomelanocortin (POMC) neurons of the hypothalamus regulate feeding behavior and energy homeostasis. An understanding of the molecular signaling pathways that regulate Agrp/NPY and POMC function could lead to novel treatments for metabolic disorders. Target of Rapamycin Complex 1 (TORC1) is a nutrient-activated protein kinase and central controller of growth and metabolism. We therefore investigated the role of mammalian TORC1 (mTORC1) in Agrp neurons. We generated and characterized Agrp neuron-specific raptor knockout (Agrp-raptor KO) mice. Agrp-raptor KO mice displayed reduced, non-circadian expression of Agrp and NPY but normal feeding behavior and energy homeostasis on both normal and high fat diet. Thus, mTORC1 in Agrp neurons controls circadian expression of orexigenic neuropeptides but is dispensable for the regulation of feeding behavior and energy metabolism.

  15. Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their immunological properties.

    PubMed

    Beesley, K M; Francis, M J; Clarke, B E; Beesley, J E; Dopping-Hepenstal, P J; Clare, J J; Brown, F; Romanos, M A

    1990-07-01

    Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response. It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope. Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively. These cores could not be produced in E. coli in soluble form but were expressed to high levels in yeast. We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes. Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles. The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.

  16. Expression of gastrin-releasing peptide is increased by prolonged stretch of human myometrium, and antagonists of its receptor inhibit contractility.

    PubMed

    Tattersall, Mark; Cordeaux, Yolande; Charnock-Jones, D Stephen; Smith, Gordon C S

    2012-05-01

    Increased uterine stretch appears to increase the risk of preterm labour, but the mechanism is unknown. The aim of this study was to identify factors that mediate the effect of stretch on human myometrium.Myometrial explants, prepared from biopsies obtained at elective caesarean delivery, were either studied acutely, or were maintained in prolonged culture (up to 65 h) under tension with either a 0.6 g or a 2.4 g mass, and compared using in vitro contractility, whole genome array, and qRT-PCR. Tissue held at tonic stretch with the 2.4 g mass for either 24 or 65 h showed increased potassium chloride (KCl)-induced and oxytocin-induced contractility compared with that held with the 0.6 g mass. Gene array identified 62 differentially expressed transcripts after 65 h exposure to increased stretch. Two probes for gastrin-releasing peptide (GRP), a known stimulatory agonist of smooth muscle, were among the top five up-regulated by stretch (3.4-fold and 2.0-fold). Up-regulation of GRP mRNA by stretch was confirmed in a separate series of 10 samples using quantitative RT-PCR (qRT-PCR) (2.8-fold, P =0.01). GRP stimulated contractions acutely when added to freshly obtained myometrial strips in 2 out of 9 cases, but Western blot demonstrated expression of the GRP receptor in 9 out of a further 9 cases. Prolonged incubation of stretched explants in the GRP antagonists PD-176252 or RC-3095 (65 and 24 h, respectively) significantly reduced KCl- and oxytocin-induced contractility.Tonic stretch of human myometrium increases contractility and stimulates the expression of a known smooth muscle stimulatory agonist, GRP. Incubation of myometrium with GRP receptor antagonists attenuates the effect of stretch. GRP may be a target for novel therapies to reduce the risk of preterm birth in multiple pregnancy.

  17. Effects of trefoil peptide 3 on expression of TNF-alpha, TLR4, and NF-kappaB in trinitrobenzene sulphonic acid induced colitis mice.

    PubMed

    Teng, Xu; Xu, Ling-Fen; Zhou, Ping; Sun, Hong-Wei; Sun, Mei

    2009-04-01

    The trefoil factor (TFF) peptides are major secretory products of mucus cells of the gastrointestinal tract. There were evidences that administration of recombinant human TFF3 is effective in treatment of models of colitis, but the mechanism of the effects of rTFF3 is not fully understood. The main aims of this study is to evaluate effects of intraperitoneal injection recombinant human TFF3 on the expression of tumour necrosis factor alpha (TNF-alpha), toll-like receptor 4(TLR4), and nuclear factor kappaB (NF-kappaB) in trinitrobenzene sulphonic acid (TNBS) induced colitis mice. Distal colitis was induced in BALB/C mice by intracolonic administration of TNBS in ethanol. Treated with administration rhTFF3 for treatment group(5 mg/ml; approximately 0.5 mg/mouse), and normal saline for control for 5 consecutive days. Colonic damage score, tissue myeloperoxidase (MPO) activity, TLR4, NF-kappaB mRNA expression, and tissue TNF-alpha, TLR4, NF-kappaB production were determined, respectively. Once daily application of hTFF3 for 5 days after TNBS/ethanol had been injected, both microscopic and macroscopic injury and inflammatory index had been reduced compared with controls. In addition, decreased tissue TNF-alpha, TLR4, NF-kappaB production, and TLR4, NF-kappaB mRNA expression had been found. This study has shown that hTFF3 may have therapeutic potential in the treatment of inflammatory bowel disease, and one of the mechanisms may related to inhibit the TLR4/NF-kappaB signaling pathways.

  18. Oleic acid and glucose regulate glucagon-like peptide 1 receptor expression in a rat pancreatic ductal cell line

    SciTech Connect

    Zhang, Leshuai W.; McMahon Tobin, Grainne A.; Rouse, Rodney L.

    2012-10-15

    The glucagon-like peptide 1 receptor (GLP1R) plays a critical role in glucose metabolism and has become an important target for a growing class of drugs designed to treat type 2 diabetes. In vitro studies were designed to investigate the effect of the GLP1R agonist, exenatide (Ex4), in “on-target” RIN-5mF (islet) cells as well as in “off-target” AR42J (acinar) and DSL-6A/C1 (ductal) cells in a diabetic environment. Ex4 increased islet cell proliferation but did not affect acinar cells or ductal cells at relevant concentrations. A high caloric, high fat diet is a risk factor for impaired glucose tolerance and type-2 diabetes. An in vitro Oleic acid (OA) model was used to investigate the effect of Ex4 in a high calorie, high fat environment. At 0.1 and 0.4 mM, OA mildly decreased the proliferation of all pancreatic cell types. Ex4 did not potentiate the inhibitory effect of OA on cell proliferation. Akt phosphorylation in response to Ex4 was diminished in OA-treated ductal cells. GLP1R protein detected by western blot was time and concentration dependently decreased after glucose stimulation in OA-treated ductal cells. In ductal cells, OA treatment altered the intracellular localization of GLP1R and its co-localization with early endosome and recycling endosomes. Chloroquine (lysosomal inhibitor), N-acetyl-L-cysteine (reactive oxygen species scavenger) and wortmannin (a phosphatidylinositol-3-kinase inhibitor), fully or partially, rescued GLP1R protein in OA-pretreated, glucose-stimulated ductal cells. The impact of altered regulation on phenotype/function is presently unknown. However, these data suggest that GLP1R regulation in ductal cells can be altered by a high fat, high calorie environment. -- Highlights: ► Exenatide did not inhibit islet, acinar or ductal cell proliferation. ► GLP1R protein decreased after glucose stimulation in oleic acid-treated ductal cells. ► Oleic acid treatment altered localization of GLP1R with early and recycling

  19. Changes in the Expression of Calcitonin Gene-Related Peptide After Exposure to Injurious Stretch-Shortening Contractions

    PubMed Central

    Johnson, C.; Miller, G.R.; Baker, B.A.; Hollander, M.; Kashon, M.L.; Waugh, S; Krajnak, K.

    2016-01-01

    One of the factors that can result in musculoskeletal injuries, and time off work, is exposure to repetitive motion. The goal of this study was to determine if skeletal muscle injury induced by exposure to injurious stretch-shortening cycles (iSSCs), resulted in hyperalgesia in the hind limb and changes in calcitonin-gene related peptide (CGRP) immunolabeling in the dorsal root ganglia (DRG) in young and old male rats. Methods Young (3 mo) and old (30 mo) male Fisher 344 × BN F1 rats were anesthetized with isoflurane and the left hind limbs were exposed to 15 sets of 10 SSCs. Control animals were exposed to a single bout of SSCs of equal intensity. Sensitivity to mechanical stimulation was assessed using von Frey filaments prior to beginning the experiment, and on days 2 and 9 following exposure to iSSCs. Rats were euthanized one, 3 or 10 days after the exposure. The ipsilateral DRG were dissected from the L4-5 region of the spine, along with the left tibialis anterior (LTA) muscle. Results Rats exposed to iSSCs were more sensitive to mechanical stimulation than control rats 2 days after the exposure, and showed a reduction in peak force 3 days after exposure. Changes in sensitivity to pressure were not associated with increases in CGRP labeling in the DRG at 3 days. However, 9 days after exposure to iSSCs, old rats still displayed an increased sensitivity to mechanical stimulation, and this hyperalgesia was associated with an increase in CGRP immunolabeling in the DRG. Young rats exposed to iSSC did not display a change in CGRP immunolabeling and sensitivity to mechanical stimulation returned to control levels at 10 days. Conclusions These findings suggest that hyperalgesia seen shortly after exposure to iSSC is not influenced by CGRP levels. However, in cases where recovery from injury may be slower, as it is in older rats, CGRP may contribute to the maintenance of hyperalgesia. PMID:26972633

  20. Tumor-Penetrating Peptides

    PubMed Central

    Teesalu, Tambet; Sugahara, Kazuki N.; Ruoslahti, Erkki

    2013-01-01

    Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular “zip code” of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the

  1. The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival: a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction

    PubMed Central

    Ding, Bo; Gibbs, Peter E. M.; Brookes, Paul S.; Maines, Mahin D.

    2011-01-01

    HO-2 oxidizes heme to CO and biliverdin; the latter is reduced to bilirubin by biliverdin reductase (BVR). In addition, HO-2 is a redox-sensitive K/Ca2-associated protein, and BVR is an S/T/Y kinase. The two enzymes are components of cellular defense mechanisms. This is the first reporting of regulation of HO-2 by BVR and that their coordinated increase in isolated myocytes and intact heart protects against cardiotoxicity of β-adrenergic receptor activation by isoproterenol (ISO). The induction of BVR mRNA, protein, and activity and HO-2 protein was maintained for ≥96 h; increase in HO-1 was modest and transient. In isolated cardiomyocytes, experiments with cycloheximide, proteasome inhibitor MG-132, and siBVR suggested BVR-mediated stabilization of HO-2. In both models, activation of BVR offered protection against the ligand's stimulation of apoptosis. Two human BVR-based peptides known to inhibit and activate the reductase, KKRILHC281 and KYCCSRK296, respectively, were tested in the intact heart. Perfusion of the heart with the inhibitory peptide blocked ISO-mediated BVR activation and augmented apoptosis; conversely, perfusion with the activating peptide inhibited apoptosis. At the functional level, peptide-mediated inhibition of BVR was accompanied by dysfunction of the left ventricle and decrease in HO-2 protein levels. Perfusion of the organ with the activating peptide preserved the left ventricular contractile function and was accompanied by increased levels of HO-2 protein. Finding that BVR and HO-2 levels, myocyte apoptosis, and contractile function of the heart can be modulated by small human BVR-based peptides offers a promising therapeutic approach for treatment of cardiac dysfunctions.—Ding, B., Gibbs, P. E. M., Brookes, P. S., Maines, M. D. The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival; a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction

  2. Analysis of expressed sequence tags (ESTs) from avocado seed (Persea americana var. drymifolia) reveals abundant expression of the gene encoding the antimicrobial peptide snakin.

    PubMed

    Guzmán-Rodríguez, Jaquelina J; Ibarra-Laclette, Enrique; Herrera-Estrella, Luis; Ochoa-Zarzosa, Alejandra; Suárez-Rodríguez, Luis María; Rodríguez-Zapata, Luis C; Salgado-Garciglia, Rafael; Jimenez-Moraila, Beatriz; López-Meza, Joel E; López-Gómez, Rodolfo

    2013-09-01

    Avocado is one of the most important fruits in the world. Avocado "native mexicano" (Persea americana var. drymifolia) seeds are widely used in the propagation of this plant and are the primary source of rootstocks globally for a variety of avocado cultivars, such as the Hass avocado. Here, we report the isolation of 5005 ESTs from the 5' ends of P. americana var. drymifolia seed cDNA clones representing 1584 possible unigenes. These avocado seed ESTs were compared with the avocado flower EST library, and we detected several genes that are expressed either in both tissues or only in the seed. The snakin gene, which encodes an element of the innate immune response in plants, was one of those most frequently found among the seed ESTs, and this suggests that it is abundantly expressed in the avocado seed. We expressed the snakin gene in a heterologous system, namely the bovine endothelial cell line BVE-E6E7. Conditioned media from transfected BVE-E6E7 cells showed antimicrobial activity against strains of Escherichia coli and Staphylococcus aureus. This is the first study of the function of the snakin gene in plant seed tissue, and our observations suggest that this gene might play a protective role in the avocado seed. PMID:23811120

  3. Analysis of expressed sequence tags (ESTs) from avocado seed (Persea americana var. drymifolia) reveals abundant expression of the gene encoding the antimicrobial peptide snakin.

    PubMed

    Guzmán-Rodríguez, Jaquelina J; Ibarra-Laclette, Enrique; Herrera-Estrella, Luis; Ochoa-Zarzosa, Alejandra; Suárez-Rodríguez, Luis María; Rodríguez-Zapata, Luis C; Salgado-Garciglia, Rafael; Jimenez-Moraila, Beatriz; López-Meza, Joel E; López-Gómez, Rodolfo

    2013-09-01

    Avocado is one of the most important fruits in the world. Avocado "native mexicano" (Persea americana var. drymifolia) seeds are widely used in the propagation of this plant and are the primary source of rootstocks globally for a variety of avocado cultivars, such as the Hass avocado. Here, we report the isolation of 5005 ESTs from the 5' ends of P. americana var. drymifolia seed cDNA clones representing 1584 possible unigenes. These avocado seed ESTs were compared with the avocado flower EST library, and we detected several genes that are expressed either in both tissues or only in the seed. The snakin gene, which encodes an element of the innate immune response in plants, was one of those most frequently found among the seed ESTs, and this suggests that it is abundantly expressed in the avocado seed. We expressed the snakin gene in a heterologous system, namely the bovine endothelial cell line BVE-E6E7. Conditioned media from transfected BVE-E6E7 cells showed antimicrobial activity against strains of Escherichia coli and Staphylococcus aureus. This is the first study of the function of the snakin gene in plant seed tissue, and our observations suggest that this gene might play a protective role in the avocado seed.

  4. Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

    PubMed Central

    Abou-Samra, Abdul B.

    2012-01-01

    Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Short treatment with PTH (0–40 min) of LLCP-K1 cells stably expressing a wild-type (WT) or a phosphorylation-deficient (PD) PPR (WT-PPR or PD-PPR cells, respectively) results in similar activation of ERK1/2. Interestingly, PTH stimulation of ERK1/2 in the WT-PPR cells then decreases as a result of longer PTH (60 min) treatment, and inhibition of ERK1/2 by PTH is observed at 90 min. Strikingly, the PD-PPR cells exhibit prolonged ERK1/2 activation up to 90 min of PTH treatment. An ERK1/2-dependent increase in c-fos expression is observed in the PD-PPR cells. Subsequently, c-fos expression in the WT-PPR and PD-PPR cells was markedly attenuated by a specific ERK1/2 pathway inhibitor. Further investigations revealed that PTH treatment causes a robust recruitment of a green fluorescent protein-tagged β-arrestin2 (β-arrestin2-GFP) in the WT-PPR cells. In contrast, β-arrestin2 recruitment was reduced in the PD-PPR cells. Importantly, expression of a receptor phosphorylation-independent β-arrestin2 (R169E) in the PD-PPR cells restored the biphasic effect of PTH on ERK1/2 as in the WT-PPR cells. The study reports a novel role for receptor phosphorylation and β-arrestin2 in the subsequent inhibition of the ERK1/2 pathway and in control of gene expression. PMID:22414806

  5. Improved PET Imaging of uPAR Expression Using new 64Cu-labeled Cross-Bridged Peptide Ligands: Comparative in vitro and in vivo Studies

    PubMed Central

    Persson, Morten; Hosseini, Masood; Madsen, Jacob; Jørgensen, Thomas J. D.; Jensen, Knud J; Kjaer, Andreas; Ploug, Michael

    2013-01-01

    The correlation between uPAR expression, cancer cell invasion and metastases is now well-established and has prompted the development of a number of uPAR PET imaging agents, which could potentially identify cancer patients with invasive and metastatic lesions. In the present study, we synthesized and characterized two new cross-bridged 64Cu-labeled peptide conjugates for PET imaging of uPAR and performed a head-to-head comparison with the corresponding and more conventionally used DOTA conjugate. Based on in-source laser-induced reduction of chelated Cu(II) to Cu(I), we now demonstrate the following ranking with respect to the chemical inertness of their complexed Cu ions: DOTA-AE105 << CB-TE2A-AE105 < CB-TE2A-PA-AE105, which is correlated to their corresponding demetallation rate. No penalty in the uPAR receptor binding affinity of the targeting peptide was encountered by conjugation to either of the macrobicyclic chelators (IC50 ~ 5-10 nM) and high yields and radiochemical purities (>95%) were achieved in all cases by incubation at 95ºC. In vivo, they display identical tumor uptake after 1h, but differ significantly after 22 hrs, where the DOTA-AE105 uptake remains surprisingly high. Importantly, the more stable of the new uPAR PET tracers, 64Cu-CB-TE2A-PA-AE105, exhibits a significantly reduced liver uptake compared to 64Cu-DOTA-AE105 as well as 64Cu-CB-TE2A-AE105, (p<0.0001), emphasizing that our new in vitro stability measurements by mass spectrometry predicts in vivo stability in mice. Specificity of the best performing ligand, 64Cu-CB-TE2A-PA-AE105 was finally confirmed in vivo using a non-binding 64Cu-labeled peptide as control (64Cu-CB-TE2A-PA-AE105mut). This control PET-tracer revealed significantly reduced tumor uptake (p<0.0001), but identical hepatic uptake compared to its active counterpart (64Cu-CB-TE2A-PA-AE105) after 1h. In conclusion, our new approach using in-source laser-induced reduction of Cu(II)-chelated PET-ligands provides useful

  6. Regulation of plant vascular stem cells by endodermis-derived EPFL-family peptide hormones and phloem-expressed ERECTA-family receptor kinases.

    PubMed

    Uchida, Naoyuki; Tasaka, Masao

    2013-12-01

    Plant vasculatures are complex tissues consisting of (pro)cambium, phloem, and xylem. The (pro)cambium serves as vascular stem cells that produce all vascular cells. The Arabidopsis ERECTA (ER) receptor kinase is known to regulate the architecture of inflorescence stems. It was recently reported that the er mutation enhances a vascular phenotype induced by a mutation of TDR/PXY, which plays a significant role in procambial proliferation, suggesting that ER participates in vascular development. However, detailed molecular mechanisms of the ER-dependent vascular regulation are largely unknown. Here, this work found that ER and its paralogue, ER-LIKE1, were redundantly involved in procambial development of inflorescence stems. Interestingly, their activity in the phloem was sufficient for vascular regulation. Furthermore, two endodermis-derived peptide hormones, EPFL4 and EPFL6, were redundantly involved in such regulation. It has been previously reported that EPFL4 and EPFL6 act as ligands of phloem-expressed ER for stem elongation. Therefore, these findings indicate that cell-cell communication between the endodermis and the phloem plays an important role in procambial development as well as stem elongation. Interestingly, similar EPFL-ER modules control two distinct developmental events by slightly changing their components: the EPFL4/6-ER module for stem elongation and the EPFL4/6-ER/ERL1 module for vascular development.

  7. Co-dependence of genotype and dietary protein intake to affect expression on amino acid/peptide transporters in porcine skeletal muscle.

    PubMed

    Liu, Y; Kong, X; Li, F; Tan, B; Li, Y; Duan, Y; Yin, Y; He, J; Hu, C; Blachier, F; Wu, Guoyao

    2016-01-01

    A total of 96 barrows (48 pure-bred Bama mini-pigs representing fatty genotype, and 48 Landrace pigs representing lean genotype) were randomly assigned to either a low- or adequate-protein treatment diet. The experimental period commenced at 5 weeks of age and extended to the finishing period. After euthanasia, blood and skeletal muscle samples were collected from pigs at the nursery, growing, and finishing phases. Our results indicate that the concentrations of free AAs in the plasma and muscle decreased as the age of the pigs increased. In addition, a strain × growth phase interaction (P < 0.05) was observed for the free AA pool in the plasma and muscle. The low-protein diet upregulated (P < 0.05) the mRNA levels for T1R1/T1R3 involved in glutamate binding, but downregulated (P < 0.05) the mRNA levels for PAT1, PAT2, and ASCT2, which transport neutral AAs into muscles. Bama mini-pigs had higher (P < 0.05) mRNA levels for LAT1, SNAT2, and EAAC1, but a lower (P < 0.05) mRNA level for PepT1, compared with Landrace pigs. Collectively, our findings indicate that adequate provision of dietary protein plays an important role in regulating profiles of free AA pools and expression of key AA/peptide transporters/transceptors in a genotype- and tissue-specific manner.

  8. Glucocorticoids facilitate astrocytic amyloid-β peptide deposition by increasing the expression of APP and BACE1 and decreasing the expression of amyloid-β-degrading proteases.

    PubMed

    Wang, Yanyan; Li, Maoquan; Tang, Jun; Song, Min; Xu, Xueqing; Xiong, Jiaxiang; Li, Junxia; Bai, Yun

    2011-07-01

    In most cases, the molecular mechanism underlying the pathogenesis of sporadic Alzheimer's disease (AD) is unknown. Elevated basal cortisol levels in AD patients suggest that glucocorticoids (GC) may contribute to the development and/or maintenance of AD. Amyloid plaques are the hallmark of AD, and they are considered to play an early role in the AD process. However, little is known about how their formation is regulated by stress and GC. Astrocyte accumulation is one of the earliest neuropathological changes in AD. Here, we report that GC elevated amyloid-β (Aβ) production in primary cultures of astrocytes by increasing amyloid precursor protein (APP) and β-site APP-cleaving enzyme 1 gene expression. Notably, GC administered to normal, middle-aged mice promoted the expression of APP and β-site APP-cleaving enzyme 1 in astrocytes, as determined by double immunofluorescence. Additionally, confocal microscopy and ELISA revealed that GC markedly reduced Aβ degradation and clearance by astrocytes in vitro, indicating a decreased neuroprotective capacity of the astrocytes. This may have been due to the decrease of several Aβ-degrading proteases, such as insulin-degrading enzyme and matrix metalloproteinase-9. These effects occurred through the activation of GC receptors. Taken together, our results demonstrate that GC can enhance the production of Aβ, reduce its degradation in astrocytes, and provide a molecular mechanism linking stress factors to AD. Our study suggests that GC can facilitate AD pathogenesis and that reducing GC in the elderly and early AD patients would be beneficial.

  9. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  10. Expression and Distribution of Glucagon-Like Peptide-1 Receptor mRNA, Protein and Binding in the Male Nonhuman Primate (Macaca mulatta) Brain

    PubMed Central

    Heppner, Kristy M.; Kirigiti, Melissa; Secher, Anna; Paulsen, Sarah Juel; Buckingham, Rikley; Pyke, Charles; Knudsen, Lotte B.

    2015-01-01

    Glucagon-like peptide-1 (GLP-1) is released from endocrine L-cells lining the gut in response to food ingestion. However, GLP-1 is also produced in the nucleus of the solitary tract, where it acts as an anorectic neurotransmitter and key regulator of many autonomic and neuroendocrine functions. The expression and projections of GLP-1-producing neurons is highly conserved between rodent and primate brain, although a few key differences have been identified. The GLP-1 receptor (GLP-1R) has been mapped in the rodent brain, but no studies have described the distribution of GLP-1Rs in the nonhuman primate central nervous system. Here, we characterized the distribution of GLP-1R mRNA and protein in the adult macaque brain using in situ hybridization, radioligand receptor autoradiography, and immunohistochemistry with a primate specific GLP-1R antibody. Immunohistochemistry demonstrated that the GLP-1R is localized to cell bodies and fiber terminals in a very selective distribution throughout the brain. Consistent with the functional role of the GLP-1R system, we find the highest concentration of GLP-1R-immunoreactivity present in select hypothalamic and brainstem regions that regulate feeding, including the paraventricular and arcuate hypothalamic nuclei, as well as the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus. Together, our data demonstrate that GLP-1R distribution is highly conserved between rodent and primate, although a few key species differences were identified, including the amygdala, where GLP-1R expression is much higher in primate than in rodent. PMID:25380238

  11. Effects of postprandial starvation on mRNA expression of endocrine-, amino acid and peptide transporter-, and metabolic enzyme-related genes in zebrafish (Danio rerio).

    PubMed

    Tian, Juan; He, Gen; Mai, Kangsen; Liu, Chengdong

    2015-06-01

    The goal of this study was to systematically evaluate the molecular activities of endocrine-, amino acid and peptide transporters-, and metabolic enzyme-related genes in 35-day-old mixed-sex zebrafish (Danio rerio) after feeding . Zebrafish with initial body weights ranging from 9 to 11 mg were fasted for 384 h in a controlled indoor environment. Fish were sampled at 0, 3, 6, 12, 24, 48, 96, 192, and 384 h after fed. Overall, the present study results show that the regulatory mechanism that insulin-like growth factor I negative feedback regulated growth hormone is conserved in zebrafish, as it is in mammals, but that regulation of growth hormone receptors is highly intricate. Leptin and cholecystokinin are time-dependent negative feedback signals, and neuropeptide Y may be an important positive neuropeptide for food intake in zebrafish. The amino acid/carnitine transporters B(0,+) (ATB(0,+)) and broad neutral (0) amino acid transporter 1(B(0)AT1) mRNA levels measured in our study suggest that protein may be utilized during 24-96 h of fasting in zebrafish. Glutamine synthetase mRNA levels were downregulated, and glutamate dehydrogenase, alanine aminotransferase, aspartate transaminase, and trypsin mRNA levels were upregulated after longtime fasting in this study. The mRNA expression levels of fatty acid synthetase decreased significantly (P < 0.05), whereas those of lipoprotein lipase rapidly increased after 96 h of fasting. Fasting activated the expression of glucose synthesis genes when fasting for short periods of time; when fasting is prolonged, the mRNA levels of glucose breakdown enzymes and pentose phosphate shunt genes decreased. PMID:25805459

  12. Expression of calcitonin gene-related peptide, adenosine A2a receptor and adenosine A1 receptor in experiment rat migraine models

    PubMed Central

    LU, WENXIAN; LI, BIN; CHEN, JINBO; SU, YIPENG; DONG, XIAOMENG; SU, XINYANG; GAO, LIXIANG

    2016-01-01

    A migraine is a disabling neurovascular disorder characterized by a unilateral throbbing headache that lasts from 4 to 72 h. The headache is often accompanied by nausea, vomiting, phonophobia and photophobia, and may be worsened by physical exercise. The trigeminovascular system (TVS) is speculated to have an important role in migraines, although the pathophysiology of this disorder remains to be elucidated. Trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis (TNC) are important components of the TVS. Several clinical cases have provided evidence for the involvement of the brainstem in migraine initiation. Electrical stimulation of the trigeminal ganglion (ESTG) in rats can activate TVS during a migraine attack. Calcitonin gene-related peptide (CGRP) is an important vasoactive compound produced following TVS activation. Numerous studies have revealed that adenosine and its receptors have an important role in pain transmission and regulation process. However, only a few studies have examined whether adenosine A2a receptor (A2aR) and adenosine A1 receptor (A1R) are involved in migraine and nociceptive pathways. In the present study, CGRP, A2aR and A1R expression levels were detected in the TG and TNC of ESTG models through reverse transcription-quantitative polymerase chain reaction and western blot analysis. Tianshu capsule (TSC), a type of Chinese medicine, was also used in the ESTG rat models to examine its influence on the three proteins. Results demonstrated that CGRP, A2aR and A1R mediated pain transmission and the regulation process during migraine and the expression of the three proteins was regulated by TSC. PMID:26998280

  13. Changes in the Expressions of Iba1 and Calcitonin Gene-Related Peptide in Adjacent Lumbar Spinal Segments after Lumbar Disc Herniation in a Rat Model

    PubMed Central

    2015-01-01

    Lumbar disc herniation is commonly encountered in clinical practice and can induce sciatica due to mechanical and/or chemical irritation and the release of proinflammatory cytokines. However, symptoms are not confined to the affected spinal cord segment. The purpose of this study was to determine whether multisegmental molecular changes exist between adjacent lumbar spinal segments using a rat model of lumbar disc herniation. Twenty-nine male Sprague-Dawley rats were randomly assigned to either a sham-operated group (n=10) or a nucleus pulposus (NP)-exposed group (n=19). Rats in the NP-exposed group were further subdivided into a significant pain subgroup (n=12) and a no significant pain subgroup (n=7) using mechanical pain thresholds determined von Frey filaments. Immunohistochemical stainings of microglia (ionized calcium-binding adapter molecule 1; Iba1), astrocytes (glial fibrillary acidic protein; GFAP), calcitonin gene-related peptide (CGRP), and transient receptor potential vanilloid 1 (TRPV1) was performed in spinal dorsal horns and dorsal root ganglions (DRGs) at 10 days after surgery. It was found immunoreactivity for Iba1-positive microglia was higher in the L5 (P=0.004) dorsal horn and in the ipsilateral L4 (P=0.009), L6 (P=0.002), and S1 (P=0.002) dorsal horns in the NP-exposed group than in the sham-operated group. The expression of CGRP was also significantly higher in ipsilateral L3, L4, L6, and S1 segments and in L5 DRGs at 10 days after surgery in the NP-exposed group than in the sham-operated group (P<0.001). Our results indicate that lumbar disc herniation upregulates microglial activity and CGRP expression in many adjacent and ipsilateral lumbar spinal segments. PMID:26713069

  14. In Vivo Effects of Pichia Pastoris-Expressed Antimicrobial Peptide Hepcidin on the Community Composition and Metabolism Gut Microbiota of Rats

    PubMed Central

    Tian, Lanfang; Chen, Siyuan; Liu, Haiyan; Guo, Mingzhang; Xu, Wentao; He, Xiaoyun; Luo, Yunbo; Qi, Xiaozhe; Luo, Hongxia; Huang, Kunlun

    2016-01-01

    Hepcidin, one kind of antimicrobial peptides, is one of the promising alternatives to antibiotics with broad spectrum of antimicrobial activity. Hepcidins cloned from different kinds