1.
The role of ghrelin in the control of energy balance.
Source
Department of Internal Medicine and Endocrinology, Metabolic Diseases Institute, Obesity Research Center, University of Cincinnati, 2180 E. Galbraith Rd, Cincinnati, OH, 54237, USA.
Abstract
Ghrelin is the only potent orexigenic peptide in circulation. It stimulates food intake and leads to positive energy balance, adipogenesis, and body weight gain. However, the physiological significance of ghrelin in the regulation of energy homeostasis is controversial, since loss of ghrelin function in rodents does not necessarily lead to anorexia andweight loss. In this chapter, we discuss the metabolic function of ghrelin and are highlighting recent findings including the discovery and function of ghrelin-acylating enzyme ghrelin O-acyltransferase (GOAT). Based on available published data, we conclude that ghrelin is a principally important endogenous regulator of energy balance, which however may affect both food intake and systemic metabolism via independent mechanisms. Importantly, ghrelin, when acylated by GOAT, might represent a key molecular link between the sensing of consumed calories and the neuroendocrine control of energy homeostasis. Thus, agents antagonizing the action of ghrelin may have therapeutic potential in the therapy of obesity.
Fatigue biomarker index: An objective salivary measure of fatigue level.
Source
Hyperion Biotechnology, Inc., 13302 Langtry Rd., San Antonio, TX 78248, United States.
Abstract
Fatigue changed the composition of the small-molecular weight (sMW) proteome of saliva during a 10h session of moderate (70% of maximum ventilatory threshold) physical exertion. Saliva samples were collected from nine recreationally trained cyclists participating in a cross-over study designed to simulate prolonged manual labor, a military operation or wildfire-suppression work. During each hour of the study, participants performed an exercise program that included upper and lower body exercises separated by short periods of recovery. Over the course of the study, fatigue level increased as suggested by a significant increase in the participants' relative perceived exertion. The composition of the sMW proteome was investigated using reversed-phase liquid chromatography with mass-spectrometric detection. Isotopes of acetic anhydride were used for mass-specific labeling of samples and subsequent identification of ions with significant changes in intensity. Cluster analysis was used to identify a pair of peptides with concentrations that changed in opposite directions with fatigue level, i.e. concentration of one peptide increased while concentration of the other decreased. The sequences of the two peptides were determined by high-resolution mass spectrometry. The ratio of the ion intensities of these two peptides, referred to as the fatigue biomarker index, was calculated for subjects throughout the study. The FBI values from the start of the study likely arose from a different distribution than the FBI values measured at the end of the study (Mann-Whitney test, P<.05). While this study is restricted to a small population of recreationally trained cyclists performing exercise under controlled conditions, it holds promise for the development of an objective salivary measurement of fatigue that is applicable to a much broader population performing in uncontrolled environments.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Extensive manipulation of caseicin A and B highlighting the tolerance of these antimicrobial peptides to change.
Source
Microbiology Department, University College Cork, Cork, Ireland.
Abstract
Caseicin A and B are low molecular weight antimicrobial peptides which are released by proteolytic digestion of sodium caseinate. Caseicin A (IKHQGLPQE) is a nine amino acid, cationic peptide and Caseicin B (VLNENLLR) is a neutral eight amino acid peptide, both of which have previously been shown to exhibit antibacterial activity against a number of pathogens including Cronobacter sakazakii. Previously, four variants of each of caseicin which differed subtly from their natural counterparts were generated by peptide synthesis. Antimicrobial activity assays in this study revealed that the importance of a number of the residues within the peptides was dependent on the strain being targeted. Here this engineering-based approach is expanded on through the creation of a larger collection of 26 peptides which are altered in a variety of ways. The investigation highlighted the generally greater tolerance of caseicin B to change, the fact that changes have a more detrimental impact on anti-Gram negative activity and the surprising number of variants which exhibit enhanced activity against S. aureus.
Turkish scorpion Buthacus macrocentrus: General characterization of the venom and description of Bu1, a potent mammalian Na(+)-channel α-toxin.
Source
Department of Biology, Faculty of Science and Art, Eskisehir Osmangazi University, 26480 Eskisehir, Turkey; Departamento de MedicinaMolecular y Bioprocesos, Instituto de Biotecnología, UNAM, Av. Universidad, 2001, Col. Chamilpa, Apartado Postal 510-3, Cuernavaca, Morelos 62210, Mexico.
Abstract
The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200-4500 Da, and 6000-7500 Da. They very likely correspond to K(+)-channel and Na(+)-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na(+) currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country.
Copyright © 2012 Elsevier Ltd. All rights reserved.
Functional and proteomic analysis of submandibular saliva in rats exposed to chronic stress by immobilization or constant light.
Source
Cátedras de Química Biológica "A" y de Fisiología, Facultad de Odontología, Universidad Nacional de Córdoba, Argentina; Cátedra de Bioquímica y Biología Molecular, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Argentina.
Abstract
OBJECTIVE:
In this study, we have evaluated the effects of stress on functional and proteomic changes in submandibular saliva of rats.
DESIGN:
Male adult rats were divided in three groups: IMO (2h/day of immobilization for 7 days), LL (constant light during 20 days), C (unstressed controls submitted to 14h light-10h dark cycle). Body weight, food intake and the dryweight of submandibular gland were recorded. Saliva samples, collected under anaesthesia following i.p. administration of isoproterenol and pilocarpine (5mg/kg), were assayed for total proteins (TP), amylase activity and SDS-PAGE electrophoresis.
RESULTS:
Body weight, food intake and the dry weight of submandibular gland of IMO rats were lower than those of C and LL groups. The salivary volumes secreted in IMO and LL rats, were significantly higher than in controls. The TP output (μg protein/μg saliva/mg of dry tissue) and amylase activity output (AU/μg of saliva/mg of dry tissue) in IMO were significantly higher than in C and LL animals. The electrophoretic pattern of saliva proteins of LL rats, revealed the absence of a protein band of approximately 25kDa. This band was composed by the common salivary protein-1 and a prolactin-induced protein as identified by peptide mass fingerprinting.
CONCLUSIONS:
Differences in body weight and food intake between IMO and LL might be attributed to the sort and intensity of stressors stimuli. The changes in the volume of secreted saliva could be a compensatory mechanism in response to stressors. The increase of total protein in IMO rats and the absence of 25kDa proteins in LL, would suggest that the submandibular glands respond to the sympathetic nervous system stimuli induced by the stress with an increase of activity of the sympathetic nerves in IMO and a reduction in LL rats.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Pharmacokinetic Predictions for Patients with Renal Impairment: Focus onPeptides and Protein Drugs.
Source
University Hospital Heidelberg, Department of Clinical Pharmacology and Pharmacoepidemiology University Hospital Ulm, Medical Department I, Division of Nephrology.
Abstract
Aim(s): Drug dosage adjustments in renal impairment are usually based on estimated individual pharmacokinetics. The extent of pharmacokinetic changes in patients with renal impairment must be known for this estimation. If measured data are not available, an estimate based on drug elimination in urine of healthy subjects or patients with normal renal function is commonly made. This is not reliable, however, if renal drug metabolism is involved, as is presumably the case for many peptide and protein drugs. In the present study a new method to predict pharmacokinetic changes for such drugs based on molecular weight was derived. Methods: Articles reporting measured pharmacokinetics of peptideand protein drugs in patients with severe renal impairment or end-stage renal disease were identified from scientific literature, the pharmacokinetic parameter values were extracted, and a statistical data synthesis was performed. A sigmoid Emax-model was applied and fitted to the data and the prediction error was analyzed. Results: Overall, 98peptide and protein drugs were identified. Relevant pharmacokinetic data in patients with renal impairment were found for 21 of these drugs. The average drug clearance was 30% and the average prolongation in half-life was 3.1-fold for low-molecular-weight peptides or proteins. The median root squared percentage of the prediction error was 18% (drug clearance) and 12% (half-life). Conclusion: An apparently continuous non-linear relationship between molecular weightand pharmacokinetic alterations in patients with severe renal impairment was found. The derived equations could be used as a rough guide for decisions on drug dosage adjustments in such patients. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.
© 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.
Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-(TDOPA)3-flower-like gold–Fe3O4 optical nanoparticles.
Authors
Source
Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2011.
2011 Oct 06 [updated 2012 Jan 05].
Excerpt
Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degrading the basement membrane and ECM (3). Gold has not been used as an X-ray contrast agent in vivo. Gold has a higher atomic number and a higher absorption coefficient than iodine, providing 2.7-fold greater contrast/weight than iodine (4). Furthermore, imaging gold at 80–100 keV reduces interference from bone absorption and provides lower soft tissue absorption, which would reduce radiation to patients. Hainfeld et al. (4) used gold nanoparticles (AuNPs; 1.9 nm in diameter, ~50 kDa) as a computed tomography (CT) contrast agent in mice; these experiments showed enhanced CT contrast of the vasculature, kidneys, and tumors in mice. However, plasma proteins in blood adsorb onto the surface of bare AuNPs, which produces large aggregates (5) that may result in altered pharmacokinetics and biodistribution of AuNPs (6). Polyethylene glycol (PEG) is found to minimize nonspecific adsorption of proteins onto NPs and to reduce their uptake by the liver (6). PEG-AuNPs have been being studied as cancer CT imaging and photothermal agents (7). Several families of MMPs are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastases (8-11). MMP expression is highly regulated in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages. Elevated levels of MMPs have been found in tumors associated with a poor prognosis for cancer patients (12). The peptide Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-NH2 was found to be a MMP substrate and is cleaved between the Leu and Gly residues. Lee et al. (13) used this sequence with a Cy5.5 NIR dye molecule to attach to AuNPs to form fluorescence-quenched nanoparticles (Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-AuNPs (Cy5.5-MMP-AuNPs or GANPs). The Cy5.5 molecules are in close proximity, resulting in fluorescence quenching because of efficient fluorescence resonance energy transfer to Au. NIR fluorescence signal will increase when the Leu-Gly bond is cleaved by MMPs, releasing Cy5.5-containing fragments. Cy5.5 is a NIR fluorescent dye with an absorbance maximum at 675 nm, an emission maximum at 694 nm, and a high extinction coefficient of 250,000 M−1cm−1. Cy5.5-MMP-AuNPs are being developed for NIR fluorescence imaging of MMPs expressed in tumors, atherosclerosis, myocardial infarction, and other diseases. Xie et al. (14) replaced the AuNP with Au-Fe3O4, a composite NP shaped like a flower, to induce a fluorescently quenched state to the overall nanostructure. There were three iron oxide “petals” on each AuNP. The MMP peptide was covalently linked to an anchoring unit, Lys-tridihydrophenylalanine (Lys-TDOPA) on the surface of the iron oxide NP. The gold surface was passivated with a thiolated PEG (SH-PEG5000). Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-(Lys-TDOPA)3-flower-like gold–Fe3O4 optical NPs (FANPs) have been evaluated for imaging protease expression in vivo.
Sections
Targeted Delivery of Proteins into the Central Nervous System Mediated by Rabies Virus Glycoprotein-Derived Peptide.
Source
School of Pharmaceutical Sciences, Southwest University, Tian Sheng Road, Beibei District, Chongqing, 400716, China, Fuailing1008@hotmail.com.
Abstract
PURPOSE:
Delivery of therapeutic proteins across the blood-brain barrier (BBB) is severely limited by their size and biochemical properties. Here we showed that a 39-amino acid peptide derived from the rabies virus glycoprotein (RDP) was exploited as an efficient protein carrier for brain-targeting delivery.
METHODS:
Three proteins with different molecular weight and pI, β-galactosidase (β-Gal), luciferase (Luc) and brain-derived neurotrophic factor (BDNF), were fused to RDP and intravenously injected into the mice respectively. The slices of different tissues with X-Gal staining were used to examine whether RDP could deliver β-Gal targeted into the CNS. The time-course relationship of RDP-Luc was studied to confirm the transport efficiency of RDP. The neuroprotective function of RDP-BDNF was examined in mouse experimental stroke to explore the pharmacological effect of RDP fusion protein.
RESULTS:
The results showed that the fusion proteins rapidly and specific entered the nerve cells in 15 min, and the t(1/2) was about 1 hr. Furthermore, RDP-BDNF fusion protein showed the neuroprotective properties in mouse experimental stroke including reduction of stroke volume and neural deficit.
CONCLUSIONS:
RDP provides an effective approach for the targeted delivery of biological active proteins into the central nervous system.
Critical issues related to transfersomes - novel vesicular system.
Source
HOD, Department of Pharmaceutics, Nalanda College of Pharmacy, India.
Abstract
It has become increasingly apparent that vesicular drug delivery elicits modest possessions in drug targeting. Transfersomes are a form of elastic or deformable vesicle, which were first introduced in the early 1990s. Elas ticity can be achieved by using an edge activator in the lipid bilayer structure. Molecules greater than 500 Da normally do not cross the skin. This prevents epicutaneous delivery of the high molecular weight therapeutics as well as non-invasive transcutaneous immunisation. Transdermal route will always remain a lucrative area for drug delivery. With the advent of new categories of drugs like peptides this route has captured more focus to combat the problems related to their delivery through oral route. But the transdermal route is equally filled with the hopes and disappointments as the transport of drug through this route faces many problems especially for the large molecules. To answer this problem many approaches were adopted. One of the very recent approaches is the use of ultra-deformable carrier systems (transfersomes). They have been used as drug carriers for a range of small molecules, peptides, proteins and vaccines, both in vitro and in vivo. Transfersomes penetrate through the pores of stratum corneum which are smaller than its size and get into the underlying viable skin in intact form. This is because of its deformable nature. The aim of this article is explanation the formation of micelle and vesicles, various types of vesicles, specifically focusing on transfersomes.
- PMID:
- 22230977
- [PubMed - in process]
Short peptide tools for monitoring caspase and proteasome activities in embryonal and adult rat brain lysates: an approach for the differential identification of proteases.
Source
A. A. Bogomoletz Institute of Physiology, National Academy of Science of Ukraine, 01024, Kiev, Bogomoletz str., 4; Department of Neurochemistry.
Abstract
The numerous caspase-like activities present in nervous tissue can be investigated with labeled peptides. However, the cross-reactivities of peptides with both proteasomes and caspases complicate the analysis of protease activity. The pharmacological features of substrates and inhibitors specific for either caspases or proteasome caspase-like proteases in rat brain lysates were similar or identical to the profiles of commercially purified proteasome preparations. Caspase inhibitors bind directly to active proteasome centers, thus competing with selective antagonists of proteasomes. Separation of lysates by molecular weight does not separate active caspases from proteasomes because these enzymes co-localize under native electrophoresis. The addition of ATP or its analogs is associated with the differential modulation of proteasomal activity, which also leads to ambiguity in the data. However, induced caspase activity could be successfully differentiated from proteasome activity in embryonal brain lysates with the non-selective caspase inhibitors Z-VAD-FMK and Q-VD-OPh and the proteasome inhibitor AdaAhx(3)L(3)VS, which are not cross-reactive. This strategy is proposed for the simultaneous examination of caspases and proteasomes using proteolysis experiments. The present study reveals that all of the caspase-like activities in the tissue lysates of non-injured adult rat brains were related to proteasomal caspase-like activities.
A novel extract from bovine colostrum whey supports anti-bacterial and anti-viral innate immune functions in vitro and in vivo I. Enhanced immune activity in vitro translates to improved microbial clearance in animal infection models.
Source
NIS Labs, Klamath Falls, OR 97601, United States.
Abstract
OBJECTIVE:
To evaluate effects on the innate immune system after exposure to, a consumable low-molecular weightfraction (CLMWF) of immunoglobulin-depleted bovine colostrum whey.
METHODOLOGY:
Cell-based immune assays were performed in vitro, and host resistance towards bacterial and viral infection was evaluated in two mouse studies.
RESULTS:
In vitro data showed a multimodal effect, as CLMWF-treatment resulted in rapid increase in phagocytosis. CLMWF increased chemotaxis of polymorphonuclear cells towards the bacterial peptide f-MLP. CLMWF treatment of natural killer cells increased expression of the CD69 activation marker. Mononuclear phagocytes showed decreased numbers of CD14(bright) and increased number of CD14(dim) cells. The remaining CD14(bright) cells showed reduced expression of CD80 and CD86, whereas CD14(dim) cells showed increased expression of CD80 and CD86, suggesting dendritic cell maturation. Mouse models were applied to evaluate the immune-modulating capacity of CLMWF when consumed acutely during bacterial (Steptococcus) and viral (Influenza) infections in vivo. Reduced bacterial and viral loads were observed in lungs within 24h. Viral load was also reduced when CLMWF was introduced intranasally.
CONCLUSION:
The data suggest that the support of antimicrobial immune defense mechanisms and maturation of antigen-presenting cells in vitro translates to protection in vivo when product is introduced across mucosal membranes.
Copyright © 2011. Published by Elsevier Inc.
Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens.
Source
Centre of Competence for the Innovation in the Agro-environmental Sector, University of Torino, via L. da Vinci 44, I-10095 Grugliasco (TO), Italy.
Abstract
An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351bp contained two introns. One intron was of 54bp, while the other was of 50bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.
Copyright © 2011 Elsevier B.V. All rights reserved.
Antioxidant activity of protein hydrolysates from aqueous extract of velvet antler (Cervus elaphus) as influenced by molecular weight and enzymes.
Source
Beijing Higher Institution Engineering Research Center of Food Additives and Ingredient, Beijing Technology and Business University, Beijing 100048, China.
Abstract
The crude protein hydrolysates from aqueous extract of velvet antler (AEVA) were prepared by simulated gastrointestinal digestion (SGI, pepsin-pancreatin) using pancreatin-pepsin, alcalase and neutrase. The resulting hydrolysates were separated by sequential ultrafiltration into four fractions. The antioxidant activities of peptide fractions were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging and Fe(2+)-chelating assays. Results showed that the hydrolysate prepared by SGI had a low degree of hydrolysis, which was significantly improved with altered proteases, such as pancreatin-pepsin and alcalase. Antioxidant activities of peptide fractions varied with molecular weight (MW) and the enzyme used. Generally, low-MW peptide fractions had higher ABTS radical scavenging activity and Fe(2+)-chelating ability, and high-MW peptide fractions were more effective in DPPH radical scavenging activity and reducing power.
- PMID:
- 22224289
- [PubMed - in process]
Characterization of oxidation products from 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine in aqueous solutions and their reactions with cysteine, histidine and lysine residues.
Source
Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany; Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany; LIFE - Leipzig Research Center for Civilization Diseases, Universität Leipzig, Germany.
Abstract
This report focuses on studies of lipid peroxidation products reactivity towards the side chains of cysteine, histidine, and lysine residues in structurally unordered peptides. Thus we have analyzed linoleic acid peroxidation products (LaPP) obtained by incubating 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine (PLPC) overnight with or without H(2)O(2) in the presence or absence of CuCl. In total, 55 different LaPP were identified with 26 containing reactive carbonyl groups. The strongest oxidation conditions (H(2)O(2) and Cu(I), i.e. a Fenton-like reagent) yielded 51 LaPP, whereas air oxidation produced only 12 LaPP. Independent of the oxidation conditions, around half of all LaPP were short-chain (oxidative cleavage) and the others long-chain (oxygen addition) PLPC oxidation products. The stronger oxidation conditions increased the number of LaPP, but also oxidized the added peptide Ac-PAAPAAPAPAEXTPV-OH (X=Cys, His or Lys) very quickly, especially under Fenton conditions. Thus, PLPC was oxidized by milder conditions (air or Cu(I)), incubated with the peptide and the peptide modifications were then analyzed by nano-RPC-ESI-Orbitrap-MS. Ten LaPP-derived peptide modifications were identified at lysine, whereas nine products were identified for cysteine and only three for histidine. Three high molecular weight LaPP still esterified to the GPC backbone were detected on Lys-containing peptide. Furthermore, three LaPP-derived mass shifts were obtained at cysteine, which have not previously been reported.
Copyright © 2012. Published by Elsevier Ireland Ltd.
Functional protease profiling for diagnosis of malignant disease.
Source
Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, Heidelberg, Germany. peter.findeisen@ikc.ma.uni-heidelberg.de.
Abstract
Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches.
Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22213637
- [PubMed - as supplied by publisher]
Molecular characterization and tissue expression profile of three novel ovine genes: ATP5O, NDUFA12 and UQCRH from muscle full-length cDNA library of black-boned sheep.
Source
Yunnan Key Laboratory of Animal Nutrition and Feed Science, Yunnan Agricultural University, Kunming, 650201, China.
Abstract
Three novel ovine genes were obtained from muscle full-length cDNA library of black-boned sheep. Sequence analysis revealed that nucleotide sequences of these genes were not homologous to any of the known sheep or goat genes, but these genes have high similarity to ATP synthase subunit O (ATP5O), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 (NDUFA12) and ubiquinol-cytochrome c reductase hinge protein (UQCRH) genes of other mammal animals (accession number: FJ546085, FJ546078 and FJ546083). The alignment analysis showed that the ovine ATP5O, NDUFA12 and UQCRH genes and proteins have closer genetic relationships with the ATP5O, NDUFA12 and UQCRH genes and proteins from cattle. Conserved domain prediction showed that these three genes included OSCP, NDUFA12 superfamily and UCR-hinge superfamily domains respectively. The deduced sequence of ATP5O, NDUFA12 and UQCRH protein had 213, 145 and 91 amino acid residues, with a molecular weight of approximately 23419.66, 17089.50 and 10657.75 Da and a theoretical isoelectric point of 9.90, 9.68 and 4.45. The secondary structure prediction revealed that 60% helix structure in ATP5O, 60% coils in NDUFA12 and no strand in UQCRH. One potential signalpeptide structure in ATP5O protein were found. NDUFA12 and UQCRH have the extremely low possibility of signalpeptides. Meanwhile, RasMol was used for visualizing the PDB files generated by Swiss-Model in cartoon or three-dimensional format. ATP5O and UQCRH protein were modeled by Swiss-Model. Tissue expression profile indicated that the ovine ATP5O, NDUFA12 and UQCRH genes could be expressed in all detected tissues including muscles, heart, liver, spleen, lung, kidney and adipose tissues, but the expression abundance of these genes were various in the different tissues. Our experiment supplied the primary foundation for further researches on these three ovine genes.
Inhibition of the STAT3 signaling pathway is involved in the antitumor activity of cepharanthine in SaOS2 cells.
Source
Institute of Vascular Medicine, Medical Research Center, Peking University Third Hospital, and Key Laboratory of Cardiovascular MolecularBiology and Regulatory Peptides, Ministry of Health, Beijing 100191, China.
Abstract
Aim:To investigate the molecular mechanisms underlying the antitumor activity of cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata.Methods:Human osteosarcoma cell line SaOS2 was used. MTT assay, Hoechst 33342 nuclear staining, flow cytometry, Western blotting and nude mouse xenografts of SaOS2 cells were applied to examine the antitumor activity of CEP in vitro and in vivo. The expression levels of STAT3 and its downstream signaling molecules were measured with Western blotting and immunochemistry analysis. The activity of STAT3 was detected based on the phosphorylation level of STAT3, luciferase gene reporter assay and translocation of STAT3 to the nucleus.Results:Treatment of SaOS2 cells with CEP (2.5-20 μmol/L) inhibited the cell growth in a concentration- and time-dependent manner. CEP (10 μmol/L) caused cell cycle arrest at G(1) phase and induced apoptosis of SaOS2 cells. CEP (10 and 15 μmol/L) significantly decreased the expression of STAT3 in SaOS2 cells. Furthermore, CEP (5 and 10 μmol/L) significantly inhibited the expression of target genes of STAT3, including the anti-apoptotic gene Bcl-xL and the cell cycle regulators c-Myc and cyclin D1. In nude mouse xenografts of SaOS2 cells, CEP (20 mg·kg(-1)·d(-1), ip for 19 d) significantly reduced the volume and weight of the tumor.Conclusion:Our findings suggest that inhibition of STAT3 signaling pathway is involved in the anti-tumor activity of CEP.
Applications of saturation transfer difference NMR in biological systems.
Source
Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Abstract
The method of saturation transfer difference (STD) nuclear magnetic resonance (NMR) is an indispensable NMR tool in drug discovery. It identifies binding epitope(s) at the atomic resolution of small molecule ligands (e.g. organic drugs,peptides and oligosaccharides), while interacting with their receptors, such as proteins and/or nucleic acids. The method is widely used to screen active drug molecules, simultaneously ranking them in a qualitative way. STD NMR is highly successful for a variety of high molecular weight systems, such as whole viruses, platelets, intact cells, lipopolysaccharide micelles, membrane proteins, recombinant proteins and dispersion pigments. Modifications of STD pulse programs using (13)C and (15)N nuclei are now used to overcome the signal overlapping that occurs with more complex structures.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Possible import routes of proteins into the cyanobacterial endosymbionts/plastids of Paulinella chromatophora.
Source
Department of Genomics, Faculty of Biotechnology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-148, Wrocław, Poland, pamac@smorfland.uni.wroc.pl.
Abstract
The rhizarian amoeba Paulinella chromatophora harbors two photosynthetically active and deeply integrated cyanobacterial endosymbionts acquired ~60 million years ago. Recent genomic analyses of P. chromatophora have revealed the loss of many essential genes from the endosymbiont's genome, and have identified more than 30 genes that have been transferred to the host cell's nucleus through endosymbiotic gene transfer (EGT). This indicates that, similar to classical primary plastids, Paulinella endosymbionts have evolved a transport system to import their nuclear-encoded proteins. To deduce how these proteins are transported, we searched for potential targeting signals in genes for 10 EGT-derived proteins. Our analyses indicate that five proteins carry potential signal peptides, implying they are targeted via the host endomembrane system. One sequence encodes a mitochondrial-like transit peptide, which suggests an import pathway involving a channel protein residing in the outer membrane of the endosymbiont. No N-terminal targeting signals were identified in the four other genes, but their encoded proteins could utilize non-classical targeting signals contained internally or in C-terminal regions. Several amino acids more often found in the Paulinella EGT-derived proteins than in their ancestral set (proteins still encoded in the endosymbiont genome) could constitute such signals. Characteristic features of the EGT-derived proteins are low molecular weight and nearly neutral charge, which both could be adaptations to enhance passage through the peptidoglycan wall present in the intermembrane space of the endosymbiont's envelope. Our results suggest that Paulinella endosymbionts/plastids have evolved several different import routes, as has been shown in classical primary plastids.
- PMID:
- 22209953
- [PubMed - as supplied by publisher]
Crotamine, a small basic polypeptide myotoxin from rattlesnake venom with cell-penetrating properties.
Source
Institute of Marine Sciences, Federal University of Ceará, Fortaleza, CE 60165-081, Brazil. gandhi.radis@ufc.br.
Abstract
Crotamine, a low molecular weight cationic polypeptide from the venom of the South American rattlesnake Crotalus durissus terrificus is a natural cell-penetrating peptide with functional versatility. The presence of nine lysine residues and three disulfide bonds renders crotamine highly compact, stable and positively charged. Topologically, crotamine adopts an ancient β-defensin fold that is found in diverse families of endogenous and venom polypeptides dedicated to host defense. Crotamine is unique among several classes of bioactive peptides because it possesses both cell penetrating and antimicrobial activities and selective biological action toward some cell types at a given cell cycle phase. Because it can rapidly and efficiently translocate into actively proliferating cells, crotamine is being investigated for labeling highly replicating cells and for use as a chemotherapeutic adjuvant. Peptides derived from crotamine, nucleolar targeting peptides (NrTPs), have been designed and are being studied. NrTPs retain some crotamine properties, such as efficient cellular uptake and preferential nuclear localization whereas they improve upon other properties. For example, NrTPs are smaller than crotamine, show higher preferential nucleolar localization, and better facilitate ZIP-code localization of therapeutic proteins.
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