ABSTRACT
Post-translational modifications (PTMs) diversify peptide structure and allow for greater flexibility within signaling networks. The cardiac neuromuscular system of the American lobster, Homarus americanus, is made up of a central pattern generator, the cardiac ganglion (CG), and peripheral cardiac muscle. Together, these components produce flexible output in response to peptidergic modulation. Here, we examined the role of PTMs in determining the effects of a cardioactive neuropeptide, myosuppressin (pQDLDHVFLRFamide), on the whole heart, the neuromuscular junction/muscle, the isolated CG, and the neurons of the CG. Mature myosuppressin and noncyclized myosuppressin (QDLDHVFLRFamide) elicited similar and significant changes in whole heart contraction amplitude and frequency, stimulated muscle contraction amplitude and the bursting pattern of the intact and ligatured neurons of the ganglion. In the whole heart, nonamidated myosuppressin (pQDLDHVFLRFG) elicited only a small decrease in frequency and amplitude. In the absence of motor neuron input, nonamidated myosuppressin did not cause any significant changes in the amplitude of stimulated contractions. In the intact CG, nonamidated myosuppressin elicited a small but significant decrease in burst duration. Further analysis revealed a correlation between the extent of modulation elicited by nonamidated myosuppressin in the whole heart and the isolated, intact CG. When the neurons of the CG were physically decoupled, nonamidated myosuppressin elicited highly variable responses. Taken together, these data suggest that amidation, but not cyclization, is critical in enabling this peptide to exert its effects on the cardiac neuromuscular system.NEW & NOTEWORTHY Myosuppressin (pQDLDHVFLRFamide), a well-characterized crustacean neuropeptide, and its noncyclized (QDLDHVFLRFamide) and nonamidated (pQDLDHVFLRFG) isoforms alter the output of the cardiac neuromuscular system of the American lobster, Homarus americanus. Mature myosuppressin and noncyclized myosuppressin elicited similar and significant changes across all levels of the isolated system, whereas responses to nonamidated myosuppressin were significantly different from other isoforms and were highly variable. These data support the diversity of peptide action as a function of peptide structure.
Subject(s)
Nephropidae , Neuropeptides , Animals , Heart/physiology , Muscles , Nephropidae/physiology , Neuropeptides/pharmacology , Protein Isoforms/pharmacologyABSTRACT
The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.
Subject(s)
Feeding Behavior/drug effects , Glucose/metabolism , Protein Isoforms/pharmacology , Ribosomal Protein S6 Kinases, 90-kDa/pharmacology , Ribosomal Protein S6 Kinases/pharmacology , Agouti-Related Protein/metabolism , Animals , Eating/genetics , Hypothalamus/metabolism , Mice , Signal Transduction/genetics , TOR Serine-Threonine Kinases/metabolismABSTRACT
Phoenixin (Pnx) is an endogenous peptide known to be involved in reproduction and food intake in rats, with two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleost. Here, pnx was cloned and was detected in all tissues of both male and female in spotted scat (Scatophagus argus), including growth axis, hypothalamus, pituitary, and liver. Real-time PCR analysis showed that pnx in the hypothalamus increased significantly after 2â¯d and 7â¯d fasting, while reduced significantly after re-feeding (Pâ¯<â¯0.05). When pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20 (10â¯nM and 100â¯nM) for 6â¯h, the expression of ghrhr (growth hormone-releasing hormone receptor) and gh (growth hormone) in the pituitary, and ghr1 (growth hormone receptor 1) in the liver increased significantly, except ghr2 (growth hormone receptor 2) incubated with 10â¯nM and 100â¯nM Pnx-20 and ghr1 incubated with 10â¯nM Pnx-20. Similarly, the expression of ghrhr and gh in the pituitary, as well as ghr1 and ghr2 in the liver, increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10â¯ng/g and 100â¯ng/g body weight). These results indicate that Pnx is likely to be involved in the regulation of food intake, and also regulates the growth of S. argus by increasing ghrhr and gh expression in the pituitary, ghr1 and ghr2 in the liver, and ghr1 directly in the liver.
Subject(s)
Energy Intake , Fish Proteins/metabolism , Gene Expression Regulation, Developmental , Hypothalamic Hormones/metabolism , Hypothalamus/metabolism , Peptide Hormones/metabolism , Perciformes/physiology , Animals , Aquaculture , China , Energy Intake/drug effects , Female , Fish Proteins/administration & dosage , Fish Proteins/genetics , Fish Proteins/pharmacology , Gene Expression Regulation, Developmental/drug effects , Growth Hormone/agonists , Growth Hormone/genetics , Growth Hormone/metabolism , Hypothalamic Hormones/administration & dosage , Hypothalamic Hormones/genetics , Hypothalamic Hormones/pharmacology , Hypothalamus/drug effects , Injections, Intraperitoneal , Liver/drug effects , Liver/metabolism , Male , Organ Specificity , Peptide Hormones/administration & dosage , Peptide Hormones/genetics , Peptide Hormones/pharmacology , Perciformes/growth & development , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Protein Isoforms/administration & dosage , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Random Allocation , Receptors, Neuropeptide/agonists , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide/metabolism , Receptors, Pituitary Hormone-Regulating Hormone/agonists , Receptors, Pituitary Hormone-Regulating Hormone/genetics , Receptors, Pituitary Hormone-Regulating Hormone/metabolism , Receptors, Somatotropin/agonists , Receptors, Somatotropin/genetics , Receptors, Somatotropin/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Tissue Culture Techniques/veterinary , Weight GainABSTRACT
Many neuropeptides are members of peptide families, with multiple structurally similar isoforms frequently found even within a single species. This raises the question of whether the individual peptides serve common or distinct functions. In the accompanying paper, we found high isoform specificity in the responses of the lobster (Homarus americanus) cardiac neuromuscular system to members of the pyrokinin peptide family: only one of five crustacean isoforms showed any bioactivity in the cardiac system. Because previous studies in other species had found little isoform specificity in pyrokinin actions, we examined the effects of the same five crustacean pyrokinins on the lobster stomatogastric nervous system (STNS). In contrast to our findings in the cardiac system, the effects of the five pyrokinin isoforms on the STNS were indistinguishable: they all activated or enhanced the gastric mill motor pattern, but did not alter the pyloric pattern. These results, in combination with those from the cardiac ganglion, suggest that members of a peptide family in the same species can be both isoform specific and highly promiscuous in their modulatory capacity. The mechanisms that underlie these differences in specificity have not yet been elucidated; one possible explanation, which has yet to be tested, is the presence and differential distribution of multiple receptors for members of this peptide family.
Subject(s)
Nephropidae/drug effects , Nervous System/drug effects , Neuropeptides/pharmacology , Protein Isoforms , Animals , Digestive System/drug effects , Digestive System/innervation , Ganglia, Invertebrate/drug effects , Ganglia, Invertebrate/physiology , Muscle Contraction/drug effects , Nephropidae/physiology , Protein Isoforms/pharmacologyABSTRACT
Although the crustacean heart is modulated by a large number of peptides and amines, few of these molecules have been localized to the cardiac ganglion itself; most appear to reach the cardiac ganglion only by hormonal routes. Immunohistochemistry in the American lobster Homarus americanus indicates that pyrokinins are present not only in neuroendocrine organs (pericardial organ and sinus gland), but also in the cardiac ganglion itself, where pyrokinin-positive terminals were found in the pacemaker cell region, as well as surrounding the motor neurons. Surprisingly, the single pyrokinin peptide identified from H. americanus, FSPRLamide, which consists solely of the conserved FXPRLamide residues that characterize pyrokinins, did not alter the activity of the cardiac neuromuscular system. However, a pyrokinin from the shrimp Litopenaeus vannamei [ADFAFNPRLamide, also known as Penaeus vannamei pyrokinin 2 (PevPK2)] increased both the frequency and amplitude of heart contractions when perfused through the isolated whole heart. None of the other crustacean pyrokinins tested (another from L. vannamei and two from the crab Cancer borealis) had any effect on the lobster heart. Similarly, altering the PevPK2 sequence either by truncation or by the substitution of single amino acids resulted in much lower or no activity in all cases; only the conservative substitution of serine for alanine at position 1 resulted in any activity on the heart. Thus, in contrast to other systems (cockroach and crab) in which all tested pyrokinins elicit similar bioactivities, activation of the pyrokinin receptor in the lobster heart appears to be highly isoform specific.
Subject(s)
Heart/drug effects , Myocardial Contraction/drug effects , Nephropidae/physiology , Neuropeptides/pharmacology , Amino Acid Sequence , Animals , Ganglia, Invertebrate/physiology , Heart/innervation , Neuropeptides/physiology , Protein Isoforms/pharmacology , Protein Isoforms/physiologyABSTRACT
This study was designed to investigate the antimicrobial activity of two synthetic antimicrobial peptides from an aquatic organism, tilapia piscidin 3 (TP3) and tilapia piscidin 4 (TP4), in vitro and in a murine sepsis model, as compared with ampicillin, tigecycline, and imipenem. Mice were infected with (NDM-1)-producing K. pneumonia and multi-drug resistant Acinetobacter baumannii, and subsequently treated with TP3, TP4, or antibiotics for different periods of time (up to 168 h). Mouse survival and bacterial colony forming units (CFU) in various organs were measured after each treatment. Toxicity was determined based on observation of behavior and measurement of biochemical parameters. TP3 and TP4 exhibited strong activity against K. pneumonia and A. baumannii in vitro. Administration of TP3 (150 µg/mouse) or TP4 (50 µg/mouse) 30 min after infection with K. pneumonia or A. baumannii significantly increased survival in mice. TP4 was more effective than tigecycline at reducing CFU counts in several organs. TP3 and TP4 were shown to be non-toxic, and did not affect mouse behavior. TP3 and TP4 are able at potentiate anti-Acinetobacter baumannii or anti-Klebsiella pneumonia drug activity, reduce bacterial load, and prevent drug resistance, indicating their potential for use in combating multidrug-resistant bacteria.
Subject(s)
Acinetobacter Infections/drug therapy , Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/therapeutic use , Antimicrobial Cationic Peptides/therapeutic use , Drug Resistance, Bacterial , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/drug effects , Acinetobacter Infections/microbiology , Animals , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/adverse effects , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/pharmacology , Bacterial Proteins/biosynthesis , Behavior, Animal/drug effects , Carbapenems/pharmacology , Carbapenems/therapeutic use , Drug Resistance, Multiple, Bacterial , Fish Proteins/adverse effects , Fish Proteins/genetics , Fish Proteins/pharmacology , Fish Proteins/therapeutic use , Klebsiella Infections/microbiology , Klebsiella pneumoniae/metabolism , Male , Mice, Inbred C57BL , Microbial Sensitivity Tests , Protein Isoforms/adverse effects , Protein Isoforms/genetics , Protein Isoforms/pharmacology , Protein Isoforms/therapeutic use , Recombinant Proteins/adverse effects , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Sepsis/drug therapy , Sepsis/microbiology , Survival Analysis , Tilapia , beta-Lactamases/biosynthesisABSTRACT
Iso-suillin, a natural product isolated from Suillus luteus, has been shown to inhibit the growth of some cancer cell lines. However, the molecular mechanisms of action of this compound are poorly understood. The purpose of this study was to investigate how iso-suillin inhibits proliferation and induces apoptosis in a human hepatoma cell line (SMMC-7721). We demonstrated the effects of iso-suillin on cell proliferation and apoptosis in SMMC-7721 cells, with no apparent toxicity in normal human lymphocytes, using colony formation assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Western blotting was used to examine the expression of G1 phase-regulated and apoptosis-associated protein levels in iso-suillin treated SMMC-7721 cells. The results indicated that iso-suillin significantly decreased viability, induced G1 phase arrest and triggered apoptosis in SMMC-7721cells. Taken together, these results suggest the potential of iso-suillin as a candidate for liver cancer treatment.
Subject(s)
Agaricales/metabolism , Apoptosis/drug effects , Carcinoma, Hepatocellular/drug therapy , Diterpenes/pharmacology , G1 Phase Cell Cycle Checkpoints/drug effects , Phenols/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Evaluation, Preclinical , Humans , Liver Neoplasms/drug therapy , Lymphocytes/drug effects , Membrane Potential, Mitochondrial/drug effects , Protein Isoforms/pharmacologyABSTRACT
BACKGROUND: Myocardial fibrosis is a key process in diabetic cardiomyopathy. However, their underlying mechanisms have not been elucidated, leading to a lack of therapy. The glucagon-like peptide-1 (GLP-1) enhancer, sitagliptin, reduces hyperglycemia but may also trigger direct effects on the heart. METHODS: Goto-Kakizaki (GK) rats developed type-II diabetes and received sitagliptin, an anti-hyperglycemic drug (metformin) or vehicle (n=10, each). After cardiac structure and function assessment, plasma and left ventricles were isolated for biochemical studies. Cultured cardiomyocytes and fibroblasts were used for in vitro assays. RESULTS: Untreated GK rats exhibited hyperglycemia, hyperlipidemia, plasma GLP-1 decrease, and cardiac cell-death, hypertrophy, fibrosis and prolonged deceleration time. Moreover, cardiac pro-apoptotic/necrotic, hypertrophic and fibrotic factors were up-regulated. Importantly, both sitagliptin and metformin lessened all these parameters. In cultured cardiomyocytes and cardiac fibroblasts, high-concentration of palmitate or glucose induced cell-death, hypertrophy and fibrosis. Interestingly, GLP-1 and its insulinotropic-inactive metabolite, GLP-1(9-36), alleviated these responses. In addition, despite a specific GLP-1 receptor was only detected in cardiomyocytes, GLP-1 isoforms attenuated the pro-fibrotic expression in cardiomyocytes and fibroblasts. In addition, GLP-1 receptor signalling may be linked to PPARδ activation, and metformin may also exhibit anti-apoptotic/necrotic and anti-fibrotic direct effects in cardiac cells. CONCLUSIONS: Sitagliptin, via GLP-1 stabilization, promoted cardioprotection in type-II diabetic hearts primarily by limiting hyperglycemia e hyperlipidemia. However, GLP-1 and GLP-1(9-36) promoted survival and anti-hypertrophic/fibrotic effects on cultured cardiac cells, suggesting cell-autonomous cardioprotective actions.
Subject(s)
Cardiotonic Agents/pharmacology , Diabetes Mellitus, Type 2/complications , Diabetic Cardiomyopathies/drug therapy , Glucagon-Like Peptide 1/physiology , Insulin/physiology , Pyrazines/pharmacology , Triazoles/pharmacology , Animals , Apoptosis , Cardiomegaly/drug therapy , Cardiomegaly/etiology , Cardiomegaly/metabolism , Cardiotonic Agents/therapeutic use , Cells, Cultured , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Diabetic Cardiomyopathies/metabolism , Disease Models, Animal , Drug Evaluation, Preclinical , Fibroblasts/physiology , Fibronectins/metabolism , Fibrosis , Glucagon-Like Peptide 1/pharmacology , Glucose Intolerance/drug therapy , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/therapeutic use , Male , Metformin/pharmacology , Metformin/therapeutic use , Myocardium/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , PPAR delta/metabolism , Protein Isoforms/pharmacology , Protein Isoforms/physiology , Pyrazines/therapeutic use , Rats , Sitagliptin Phosphate , Triazoles/therapeutic useABSTRACT
Accelerated atherosclerosis is the primary cardiovascular manifestation of diabetes and correlates inversely with levels of circulating adiponectin, an anti-atherosclerotic adipokine that declines in diabetes. We therefore initiated a study to examine the mechanisms by which adiponectin, a hormone released from adipose tissue, influences the proliferation of vascular smooth muscle cells (SMCs). Addition of adiponectin to quiescent porcine coronary artery SMCs increased both protein and DNA synthesis and concurrently activated ERK1/2 and Akt. By contrast, globular adiponectin, a truncated form of this protein, exhibited anti-mitogenic properties as indicated by the inhibition of protein and DNA synthesis in SMCs stimulated with platelet-derived growth factor (PDGF). Whereas globular adiponectin did not stimulate growth-related signal transduction pathways, it was able to block the PDGF-dependent phosphorylation of eukaryotic elongation factor 2 kinase, a regulator of protein synthesis. Proteolysis of adiponectin with trypsin, which produces globular adiponectin, reversed the growth-stimulating actions of the undigested protein. As the existence of globular adiponectin remains controversial, western blotting was used to establish its presence in rat serum. We found that globular adiponectin was detectable in rat serum, but this result was not obtained with all antibodies. The contrasting properties of adiponectin and its globular form with respect to SMC proliferation suggest that protection against atherosclerosis may therefore be mediated, in part, by the level of globular adiponectin.
Subject(s)
Adiponectin/chemistry , Adiponectin/metabolism , Adiponectin/pharmacology , Cell Proliferation/drug effects , Myocytes, Smooth Muscle/drug effects , Protein Folding , Adenylate Kinase/metabolism , Adiponectin/blood , Animals , Cells, Cultured , Down-Regulation/drug effects , Drug Evaluation, Preclinical , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/physiology , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Proteolysis/drug effects , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , SwineABSTRACT
Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.
Subject(s)
Bacterial Adhesion/drug effects , Staphylococcus/drug effects , Transferrins/pharmacology , Biofilms/drug effects , Dose-Response Relationship, Drug , HeLa Cells , Humans , Protein Isoforms/pharmacology , Staphylococcus/physiologyABSTRACT
UNLABELLED: Chan (Hyptis suaveolens L.) seeds have been used as food as well as in traditional medicine in several countries of America, Asia and Africa. Chan seed protein content was 13.9% on dry weight basis. Analysis of its protein composition showed 39% globulins, 36% glutelins, 24% albumins, and 1% prolamins. By defatting the flour with chloroform/methanol, it increased the extracted proteins and improved the protein band resolution after SDS-PAGE, showing 5 albumin bands, 8 globulin bands, and 2 prolamin and glutelin bands. The aromatic amino acid content in chan seeds is higher than those of other grains including maize, with good levels of branched chain amino acids. In general, except for lysine, it has a well-balanced amino acid composition, providing a good supply of almost all the essential amino acids for the different age groups. Magnesium content was high, whereas calcium, potassium, and phosphorous were in the average range when compared to barley, oat, rice, and wheat. The present results indicate that seeds from the chan plant could be relevant because of their nutritional properties and they have the potential to be widely used in the production of high-quality food. PRACTICAL APPLICATION: Chan seeds are presently used in a very limited manner as a food source; however, considering their high quality composition, they have the potential for a more extended use in the food industry.
Subject(s)
Hyptis/chemistry , Minerals/analysis , Seed Storage Proteins/analysis , Seeds/chemistry , Adult , Albumins/analysis , Albumins/chemistry , Albumins/pharmacology , Amino Acids, Essential/analysis , Child , Dietary Proteins/analysis , Dietary Proteins/pharmacology , Female , Globulins/analysis , Globulins/chemistry , Globulins/pharmacology , Humans , Infant , Lactation , Magnesium/analysis , Male , Molecular Weight , Nutritive Value , Pregnancy , Prolamins/analysis , Prolamins/chemistry , Prolamins/pharmacology , Protein Isoforms/analysis , Protein Isoforms/chemistry , Protein Isoforms/pharmacology , Seed Storage Proteins/chemistry , Seed Storage Proteins/pharmacology , Trypsin Inhibitors/analysis , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/pharmacologyABSTRACT
A major trypsin inhibitor was isolated and characterized from the seeds of the tartary buckwheat (Fagopyrum tataricum) (FtTI) by ammonium sulfate precipitation, ion exchange chromatography and centrifugal ultrafiltration. SDS-PAGE analysis under reducing condition showed that FtTI is a single polypeptide chain with a molecular mass of approximately 14kDa. The complete amino acid sequence of FtTI was established by automatic Edman degradation and mass spectrometry. It was found that the trypsin inhibitor molecule consists of 86 amino acid residues containing two disulfide bonds which connect Cys(8) to Cys(65) and Cys(49) to Cys(58). The active site of the inhibitor was found to contain an Asp(66)-Arg(67) bond. MALDI-TOF analysis showed that FtTI has two isoforms (Mr: 11.487 and 13.838kDa). Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) of 1.6nM. Analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor I family. What is more, FtTI exhibited strong inhibitory activity against phytopathogenic fungi.
Subject(s)
Antifungal Agents/chemistry , Fagopyrum/chemistry , Fungi/drug effects , Plant Proteins/chemistry , Protein Isoforms/chemistry , Seeds/chemistry , Trypsin Inhibitors/chemistry , Trypsin/metabolism , Amino Acid Sequence , Antifungal Agents/isolation & purification , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Fagopyrum/metabolism , Fungi/growth & development , Mass Spectrometry , Molecular Sequence Data , Phylogeny , Plant Diseases/microbiology , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Plant Proteins/pharmacology , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Seeds/metabolism , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Trypsin Inhibitors/isolation & purification , Trypsin Inhibitors/metabolism , Trypsin Inhibitors/pharmacology , UltrafiltrationABSTRACT
Phytocystatins are potent inhibitors of cysteine proteases and have been shown to participate in senescence, seed and organ biogenesis, and plant defense. However, phytocystatins are generally poor inhibitors of the cysteine protease, bromelain, of pineapple (Ananas comosus). Here, we demonstrated that pineapple cystatin, AcCYS1, inhibited (>95%) stem and fruit bromelain. AcCYS1 is a unique cystatin in that it contains an extended N-terminal trunk (NTT) of 63 residues rich in alanine and glutamate. A signal peptide preceding the NTT is processed in vitro by microsomal membranes giving rise to a 27-kD species. AcCYS1 mRNA was present in roots and leaves but was most abundant in fruit. Using immunofluorescence and immunoelectron microscopy with an AcCYS1-specific antiserum, AcCYS1 was found in the apoplasm. Immunoblot analysis identified a 27-kD protein in fruit, roots, and leaves and a 15-kD species in mature ripe fruit. Ripe fruit extracts proteolytically removed the NTT of 27-kD AcCYS1 in vitro to produce the 15-kD species. Mass spectrometry analysis was used to map the primary cleavage site immediately after a conserved critical glycine-94. The AE-rich NTT was required to inhibit fruit and stem bromelain (>95%), whereas its removal decreased inhibition to 20% (fruit) and 80% (stem) and increased the dissociation equilibrium constant by 1.8-fold as determined by surface plasmon resonance assays. We propose that proteolytic removal of the NTT results in the decrease of the inhibitory potency of AcCYS1 against fruit bromelain during fruit ripening to increase tissue proteolysis, softening, and degradation.
Subject(s)
Ananas/enzymology , Bromelains/antagonists & inhibitors , Cystatins/chemistry , Cystatins/metabolism , Fruit/physiology , Protein Processing, Post-Translational , Alanine , Amino Acid Sequence , Ananas/growth & development , Cystatins/genetics , Cystatins/pharmacology , Fruit/drug effects , Fruit/genetics , Glutamic Acid , Kinetics , Microsomes/drug effects , Microsomes/metabolism , Models, Biological , Molecular Sequence Data , Plant Leaves/drug effects , Plant Leaves/metabolism , Plant Roots/cytology , Plant Roots/drug effects , Plant Roots/metabolism , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Protein Processing, Post-Translational/drug effects , Protein Transport/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , Recombinant Proteins/pharmacology , Sequence AlignmentABSTRACT
In this study, we showed that WAX9D, a nonspecific lipid-transfer protein found in broccoli, binds palmitate (C16) and stearate (C18) with dissociation constants of 0.56 muM and 0.52 muM, respectively. WAX9D was fused to thioredoxin protein by genetic manipulation to enhance its solubility. The data revealed strong interaction of Trx-WAX9D with palmitate and stearate. The dissociation constants of Trx-WAX9D for palmitate and stearate were 1.1 muM and 6.4 muM, respectively. The calculated number of binding sites for palmitate and stearate was 2.5 to 2.7, indicating that Trx-WAX9D can bind three molecules of fatty acids. Additionally, Trx-WAX9D was shown to inhibit the apoptotic effect of palmitate in endothelial cells. Our data using Trx-WAX9D provide insight into the broad spectrum of its biological applications with specific palmitate binding.
Subject(s)
Brassica/chemistry , Carrier Proteins/isolation & purification , Plant Proteins/isolation & purification , Animals , Apoptosis/drug effects , Brassica/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Carrier Proteins/pharmacology , Cattle , Drug Evaluation, Preclinical , Endothelial Cells/drug effects , Endothelial Cells/physiology , Fatty Acids/metabolism , Intracellular Signaling Peptides and Proteins , Palmitic Acid/pharmacology , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Proteins/pharmacology , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , SolubilityABSTRACT
Placental human growth hormone-variant (hGH-V) and pituitary human growth hormone-N (hGH-N) are of identical size (22 kDa) but differ in 13 residues scattered throughout the protein. Several isoforms of GH are produced by the hGH-N and hGH-V genes including a 20-kDa hGH-V resulting from a 45-bp deletion caused by the use of an alternative acceptor site within exon 3. To date, the biological properties of the 20-kDa GH-V have not been characterized in vivo. Using young male Wistar rats fed either chow or a high-fat (HF) diet for 4 wk postweaning, we investigated the effect of 7 days treatment with either 22-kDa hGH-N, 20-kDa hGH-V (5 ug x g(-1) x day(-1) sc), or vehicle on body composition and endocrine and metabolic profiles. Total body growth (absolute weight gain and linear growth trajectory) in the 20-kDa hGH-V-treated animals was intermediary between that of control and hGH-N-treated animals. Both 22-kDa hGH-N and 20-kDa hGH-V significantly reduced total body fat mass compared with control animals, and there were no differences between the GH isoforms in anti-lipogenic activity in animals fed the HF diet. Fasting plasma insulin and C peptide were significantly increased in animals on the HF diet and further increased by hGH-N but were unchanged in 20-kDa hGH-V-treated animals compared with saline-treated controls. Plasma volume as assessed by hematocrit was increased in hGH-N-treated animals but was unchanged in 20-kDa hGH-V-treated animals compared with controls. Furthermore, 20-kDa hGH-V had reduced lactogenic (prolactin receptor mediated) activity characteristic of hGH-N as tested in vitro compared with the 20-kDa hGH-N and 22-kDa hGH-N variants. In summary, placental 20-kDa hGH-V retains some of the growth-promoting and all antilipogenic activities of pituitary 22-kDa hGH-N but has diminished diabetogenic and lactogenic properties compared with the native 22-kDa hGH-N.
Subject(s)
Diabetes Mellitus, Experimental/chemically induced , Growth Hormone/pharmacology , Human Growth Hormone/pharmacology , Lactation/drug effects , Lipogenesis/drug effects , Peptide Fragments/pharmacology , Placental Hormones/pharmacology , Animals , Body Weight/drug effects , Diet, Atherogenic , Drug Evaluation, Preclinical , Female , Growth Hormone/chemistry , Hypolipidemic Agents/pharmacology , Male , Molecular Weight , Placental Hormones/chemistry , Protein Isoforms/chemistry , Protein Isoforms/pharmacology , Rats , Rats, WistarABSTRACT
Conjugated linoleic acid (CLA) is a dietary chemopreventive agent that induces apoptosis in the mammary adipose vascular endothelium and decreases mammary brown adipose tissue (BAT) and white adipose tissue (WAT). To determine onset and extent of stromal remodeling, we fed CD2F1/Cr mice diets supplemented with 1 or 2 g/100 g mixed CLA isomers for 1-7 wk. BAT loss, collagen deposition, and leukocyte recruitment occurred in the mouse mammary fat pad, coincident with an increase in parenchymal-associated mast cells in mice fed both levels of CLA. Feeding experiments with purified isomers (0.5 g/100 g diet) demonstrated that these changes were induced by trans-10, cis-12 CLA (10,12-CLA), but not by cis-9, trans-11 CLA (9,11-CLA). This stromal remodeling did not require tumor necrosis factor (TNF)-alpha, a major cytokine in mast cells, as TNF-alpha null mice demonstrated collagen deposition, increased leukocytes, and BAT loss in the mammary fat pad in response to 10,12-CLA. To test the hypothesis that mast cells recruited in response to 10,12-CLA were required for stromal remodeling, Steel mice (WBB6F1/J-kit(W)/kit(W-V)), which lack functional mast cells, were examined for their stromal response to 10,12-CLA. Both wild-type and Steel mice showed a significantly increased leukocytic adipose infiltrate, collagen deposition, and decreased adipocyte size, although BAT was maintained in Steel mice. These results demonstrate that 10,12-CLA induces an inflammatory and fibrotic phenotype in the mouse mammary gland stroma that is independent of TNF-alpha or mast cells and suggest caution in the use of 10,12-CLA for breast cancer chemoprevention.
Subject(s)
Linoleic Acids, Conjugated/pharmacology , Mammary Glands, Animal/drug effects , Mammary Glands, Animal/physiology , Mast Cells/drug effects , Mast Cells/physiology , Adipose Tissue/cytology , Animals , Cell Count , Collagen/metabolism , Female , Mammary Glands, Animal/cytology , Mammary Glands, Animal/metabolism , Mast Cells/cytology , Mice , Mice, Inbred Strains , Mice, Knockout , Neutrophil Infiltration/drug effects , Protein Isoforms/pharmacology , Tumor Necrosis Factor-alpha/deficiency , Tumor Necrosis Factor-alpha/physiologyABSTRACT
Acetylcholine signaling and acetylcholinesterase (AChE) function(s) are pivotal elements in muscle development. The effects of the stimulus-dependent readthrough AChE variant, AChE-R, on leiomyomas and normal myometrium proliferation were assessed in vivo and in vitro. Histological preparations and cell cultures therefrom were obtained during hysterectomies or myomectomies and included both the leiomyoma sample and the adjacent normal uterine muscle as control. In situ hybridization procedures were performed using AChE cRNA probes complementary to the human AChE-R transcript. Antibodies against the AChE-R variant served for immunohistochemical staining. To determine the biological function of AChE-R on the uterine muscle cell cultures, we used a synthetic peptide representing the potentially cleavable morphogenically active C-terminus of AChE-R (ARP). Cell proliferation was assessed using the incorporation of 5'-bromo-2-deoxyuridine (BrDU). Leiomyomas expressed an excess of AChE-R mRNA and the AChE-R protein compared with the normal myometrium. Cell cultures originating from leiomyomas proliferated significantly faster than cultures from the adjacent myometrium (P = 0.027 at BrDU incorporation). Addition of ARP (2-200 nM) caused a dose-dependent decrease in the proliferation of cell cultures from both leiomyomas and the myometrium. The effect on the myometrium reached statistical significance (at 20 and 200 nM, P = 0.02), whereas the variability of the rapidly proliferating primary cultures was high and precluded statistical significance in the leiomyoma cultures. AChE-R is involved in the proliferation of the myometrium. The inhibitory effect of ARP on the myometrium may suggest a future therapeutic role of ARP.
Subject(s)
Acetylcholinesterase/physiology , Leiomyoma/pathology , Myometrium/cytology , Uterine Neoplasms/pathology , Acetylcholinesterase/metabolism , Acetylcholinesterase/pharmacology , Cell Proliferation , Cells, Cultured , Female , Humans , Leiomyoma/enzymology , Myometrium/enzymology , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Protein Isoforms/physiology , RNA, Messenger/analysis , Uterine Neoplasms/enzymologyABSTRACT
We have recently demonstrated in multiple independent population-based longitudinal and cross sectional analyses that the haptoglobin 2-2 genotype is associated with an increased risk for diabetic cardiovascular disease. The chief function of haptoglobin (Hp) is to bind to hemoglobin and thereby prevent hemoglobin-induced oxidative tissue damage. This antioxidant function of haptoglobin is mediated in part by the ability of haptoglobin to prevent the release of iron from hemoglobin on its binding. We hypothesized that there may be diabetes- and haptoglobin genotype-dependent differences in the amount of catalytically active redox active iron derived from hemoglobin. We tested this hypothesis using several complementary approaches both in vitro and in vivo. First, measuring redox active iron associated with haptoglobin-hemoglobin complexes in vitro, we demonstrate a marked increase in redox active iron associated with Hp 2-2-glycohemoglobin complexes. Second, we demonstrate increased oxidative stress in tissue culture cells exposed to haptoglobin 2-2-hemoglobin complexes as opposed to haptoglobin 1-1-hemoglobin complexes, which is inhibitable by desferrioxamine by either a chelation or reduction mechanism. Third, we demonstrate marked diabetes-dependent differences in the amount of redox active iron present in the plasma of mice genetically modified expressing the Hp 2 allele as compared with the Hp 1 allele. Taken together these data implicate redox active iron in the increased susceptibility of individuals with the Hp 2 allele to diabetic vascular disease.
Subject(s)
Diabetes Mellitus, Experimental/metabolism , Haptoglobins/genetics , Iron/metabolism , Oxidative Stress , Alleles , Animals , CHO Cells/drug effects , CHO Cells/metabolism , Cricetinae , Cricetulus , Deferoxamine/pharmacology , Fluoresceins/pharmacology , Genotype , Glucose/pharmacology , Glycated Hemoglobin/metabolism , Haptoglobins/chemistry , Haptoglobins/metabolism , Haptoglobins/pharmacology , Haptoglobins/physiology , Hemoglobins/metabolism , Hemoglobins/pharmacology , Humans , Iron/chemistry , Iron Chelating Agents/pharmacology , Kidney/metabolism , Lipid Peroxidation/drug effects , Mice , Models, Biological , Oxidation-Reduction , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/pharmacology , Protein Isoforms/physiology , Recombinant Fusion Proteins/physiology , Structure-Activity Relationship , TransgenesABSTRACT
We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions.
Subject(s)
Androgens/physiology , Evolution, Molecular , Guinea Pigs/genetics , Guinea Pigs/metabolism , Milk Proteins/genetics , Seminal Vesicle Secretory Proteins/genetics , Seminal Vesicle Secretory Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary , Epithelial Cells/metabolism , Male , Molecular Sequence Data , Pancreatic Elastase/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , RNA, Messenger/metabolism , Recombinant Proteins/pharmacology , Seminal Vesicle Secretory Proteins/pharmacology , Seminal Vesicles/metabolismABSTRACT
The purpose of this study is to test the hypothesis that mechanical and electrical activity in adult rat ventricular myocytes (ARVM) alters responses to proapoptotic and prosurvival ligands. The effects of electrical stimulation on myocyte survival, stress signaling, response to beta-adrenergic receptor (beta-AR)-stimulated apoptosis, and neuregulin-1beta (NRG) were examined. Electrical stimulation (6.6 V/cm; 0, 2, and 5 Hz; 2-ms duration; alternating polarity) of ARVM resulted in more than 70% capture. Although ARVM paced for 48 h showed higher mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (P < 0.05, 0 vs. 2 and 5 Hz), electrical stimulation had little effect on cell survival assessed by trypan blue uptake, CPK release, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Electrical stimulation for 24 h did not induce stress response (heat shock protein 70, 90) nor stress kinase (Erk, JNK, p38) activation. NRG stimulation of Erk and Akt was similar between paced and quiescent cells. Pacing sensitized myocytes to beta-AR-stimulated JNK phosphorylation and cell death with 0.1 microM norepinephrine (NE) in paced myocytes causing equivalent cytotoxicity to 10 microM NE in quiescent cells. NRG suppressed beta-AR-induced apoptosis through a phosphatidylinositol-3-kinase-dependent pathway in both paced and quiescent cells, although it is overwhelmed by high-NE concentration in paced cells. Thus myocyte contractility modulates both NE cytotoxicity as well as the cytoprotective effect of NRG. These results demonstrate the feasibility and importance of using electrically paced cardiomyocytes in primary culture when examining the signaling pathways of cell survival.