Asn Pro Research Articles

A supramolecular hydrogel eye-drop alleviates inflammation via clathrin-mediated endocytosis

The modulation of inflammation is effective to treat many ocular surface diseases. Thus the low bioavailability of common anti-inflammatory eye-drops urges the development of ocular drug delivery systems to extend the ocular retention and enhance the cellular uptake for improving anti-inflammatory effect of eye-drops. Here we covalently conjugate two molecules of clinically anti-inflammatory drug (i.e., dexamethasone) with a small peptide (i.e., Tyr–Glu–Asn–Pro–Thr–Tyr) to generate an anti-inflammatory hydrogel eye-drop. With a self-assembled ability, the designed supramolecular hydrogel achieves gel–sol–gel transition by varying shearing forces which increases the pre-corneal retention of drug. The fluorescent imaging reveals the efficient cellular uptake of designed conjugate via clathrin-mediated endocytosis. A rodent model of endotoxin-induced uveitis verifies that the supramolecular hydrogel eye-drop suppresses inflammation responses without ocular irritation. As a rational approach to design anti-inflammatory drugs as eye-drops, this work overcomes the frequent instillation of clinical eye-drops and further improves the bioavailability of anti-inflammatory drugs, which may provide an effective and household way to fight ocular surface inflammation.

Green Synthesis of Silver Nanoparticles Using the Tridax procumbens Plant Extract and Screening of Its Antimicrobial and Anticancer Activities.

In this study, we report the green synthesis of silver nanoparticles (AgNPs) using the aqueous leaf extract of Tridax procumbens (TNP), which acts as the source of the reducing and capping agent. The distinctive absorption at 370 nm suggested synthesis of TNPs, which was confirmed by TEM, with a size in the range of 11.1 nm to 45.4 nm and a spherical shape, having a face-centered cubic structure, analyzed by XRD, and a Zeta potential of -20.7 mV, which indicated a moderate stability of TNP. The FTIR analysis revealed the presence of amines and hydroxyl groups with fluoro compounds over the TNPs. The HRLC-MS analysis of TNPs suggested the presence of a major capping agent such as fosinopril and reducing agents such as peptides (Gln Gly Ala, Ser Pro Asn, and Leu Met), terpenoids (lupanyl acid, tiamulin), polyphenol (peucenin), and alkaloids (8′,10′-dihydroxydihydroergotamine, carteolol). The synthesized silver nanoparticles exhibited antimicrobial activity against multidrug-resistant (MDR) clinical isolates (Escherichia coli, Shigella spp., Aeromonas spp., Pseudomonas aeruginosa, and Candida tropicalis) and had anticancer activity against A459 (IC50 42.70 μg/ml). The extraction of partially purified aqueous leaf extracts by silica gel column chromatography followed by HPLC to synthesize silver nanoparticles (TNP11) and analyzed by HRLC-MS suggested that dipeptides were involved in the reduction of Ag+ to Ag0. Overall, the results showed that the green silver nanoparticles of T. procumbens could be safe, as they are endowed with potential antimicrobial activity against MDR clinical isolates and human lung carcinoma cells.

Two C-type lectins (ReCTL-1, ReCTL-2) from Rimicaris exoculata display broad nonself recognition spectrum with novel carbohydrate binding specificity

C-type lectins are Ca2+-dependent carbohydrate-binding proteins containing one or more carbohydrate-recognition domains (CRDs). C-type lectins play crucial roles in innate immunity, including nonself-recognition and pathogen elimination. In the present study, two C-type lectins (designated ReCTL-1 and ReCTL-2) were identified from the shrimp Rimicaris exoculata which dwells in deep-sea hydrothermal vents. The open reading frames of ReCTL-1 and ReCTL-2 encoded polypeptides of 171 and 166 amino acids respectively, which were both composed of a signal peptide and a single CRD. The key motifs determining the carbohydrate binding specificity of ReCTL-1 and ReCTL-2 were respectively Glu–Pro–Ala (EPA) and Gln–Pro–Asn (QPN), which were firstly discovered in R. exoculata. ReCTL-1 and ReCTL-2 displayed similar pathogen-associated molecular pattern (PAMP) binding features and they bound three PAMPs—β-glucan, lipopolysaccharide and peptidoglycan—with relatively high affinity. In addition, both could efficiently recognize and bind Gram-positive bacteria, Gram-negative bacteria and fungi. However, ReCTL-1 and ReCTL-2 exhibited different microbial agglutination activities: ReCTL-1 agglutinated Staphylococcus aureus and Saccharomyces cerevisiae, while ReCTL-2 agglutinated Micrococcus luteus, Vibrio parahaemolyticus and V. fluvialis. Both ReCTL-1 and ReCTL-2 inhibited the growth of V. fluvialis. All these results illustrated that ReCTL-1 and ReCTL-2 could function as important pattern-recognition receptors with broad nonself-recognition spectra and be involved in immune defense against invaders, but their specificities are not the same. In addition, the two ReCTLs possessed different carbohydrate binding specificities from each other and from the classical pattern: ReCTL-1 with an EPA motif bound d-galactose and l-mannose, while ReCTL-2 with a QPN motif bound d-fucose and N-acetylglucosamine.

Molecular cloning and induced expression of the plasma membrane intrinsic protein gene and promoter from mulberry (Morus multicaulis)

To isolate the plasma membrane intrinsic protein 2 (PIP2) gene and its promoter from mulberry (Morus multicaulis), and to analyse its expression under stress conditions using quantitative real-time polymerase chain reaction (qRT-PCR) analysis, the mulberry MmPIP2 gene was cloned and sequenced. The putative MmPIP2 protein was 282 amino acids long and contained Asn–Pro–Ala signature motifs. Phylogenetic analysis showed that MmPIP2 is highly conserved, exhibiting strong homology with other plant PIP2s. The 5′ flanking sequence was cloned by genome walking and numerous transcription factor binding sites were identified. qRT-PCR revealed that the expression levels of MmPIP2 differed with changes in abiotic stress, indicating that PIP2 is a multifunctional molecule in mulberry. Studying the molecular mechanisms behind adaptations to stress and strengthening stress tolerance in plants are of fundamental importance to mulberry production.

ACE Inhibitory and Antioxidant Activities of Novel Peptides from Scorpaena notata By-product Protein Hydrolysate

This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC50 values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.

Physiological Effects of Aquaporin in Regulating Drought Tolerance through Overexpressing of Festuca arundinacea Aquaporin Gene FaPIP2;1

Aquaporin (AQP) proteins serve important roles in regulating water movement across cellular membranes and affect plant responses to drought stress. The objective of this study was to characterize and examine functions of an AQP gene FaPIP2;1, isolated from a drought-tolerant perennial grass species tall fescue (Festuca arundinacea), for involvement in leaf dehydration status during water stress by overexpressing the gene in arabidopsis (Arabidopsis thaliana). FaPIP2;1 had characteristic transmembrane domains and Asn–Pro–Ala motifs and was similar to PIP2;1 in rice (Oryza sativa) and maize (Zea mays). Quantitative real-time reverse transcriptase polymerase chain reaction analysis showed that FaPIP2;1 was upregulated during moderate water stress (hydroponic culture, osmotic potential (ΨS) at −0.47 and −0.78 MPa) and the transcript level decreased as ΨS further decreased. Transgenic arabidopsis plants overexpressing FaPIP2;1 showed greater number of leaves per plant and improved survival rate compared with the wild type (WT) during drought stress. Transgenic plants also maintained higher leaf relative water content (RWC), chlorophyll content (Chl), net photosynthetic rate (Pn), and lower leaf electrolyte leakage (EL) than the WT. However, there was no difference in root length between the transgenic and WT plants following drought stress. The results demonstrated that overexpressing FaPIP2;1 could improve plant tolerance to drought stress by enhancing leaf water status, Chl, and photosynthetic rate, as well as maintaining improved cellular membrane stability relative to the WT plants. FaPIP2;1 may be used as a candidate gene for genetic modification of perennial grasses to develop new drought-tolerant germplasm and cultivars.

Isolation and structural characterization of Coryxin, a novel cyclic lipopeptide from Corynebacterium xerosis NS5 having emulsifying and anti-biofilm activity

Herein we reported the structure and several properties of a new biosurfactants produced by Corynebacterium xerosis strain NS5. This strain was capable of producing a novel lipopeptide biosurfactant that we have named coryxin. The biosurfactant structure was characterized by using Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance spectroscopy (NMR), and Liquid chromatography-mass spectrometry (LC–MS). It contained a hydrophobic moiety of 3-hydroxydecanoic acid and a peptide part predicted as a sequence of seven amino acids including Asn–Arg–Asn–Gln–Pro–Asn–Ser. Coryxin lowered the surface tension of water to 31.4mN/m, with a critical micelle concentration of 25mg/l. It was a strong emulsifier with an emulsification index of 61% against n-hexane. Coryxin showed antibacterial activity against test organisms belonging to Gram-positive and Gram-negative bacteria and disrupted preformed biofilms of Staphylococcus aureus (82.5%), Streptococcus mutans (80%), Escherichia coli (66%) and Pseudomonas aeruginosa (30%). In conclusion, microbial surfactant from C. xerosis exhibited inhibitory and disruptive activities against biofilm formation that could be of use in biofilm-related menace.

Selection of peptide ligands for piezoelectric peptide based gas sensors arrays using a virtual screening approach

Virtual and experimental affinity binding properties of 5 different peptides (cysteinylglycine, glutathione, Cys–Ile–His–Asn–Pro, Cys–Ile–Gln–Pro–Val, Cys–Arg–Gln–Val–Phe) vs. 14 volatile compounds belonging to relevant chemical classes were evaluated. The peptides were selected in order to have a large variability in physicochemical characteristics (including length).In virtual screening a rapid and cost-effective computational methodology for predicting binding scores of small peptide receptors vs. volatile compounds is proposed. Flexibility was considered for both ligands and peptides and each peptide conformer was treated as a possible receptor, generating a dedicated box and then running a docking process vs. all possible conformers of the 14 volatile compounds.The 5 peptides were covalently bound to gold nanoparticles and deposited onto 20 MHz quartz crystal microbalances to realize gas sensors. Gas sensing confirmed that each of the peptide conferred to the gold nanoparticles a particular selectivity pattern able to discriminate the 14 volatile compounds. The largest response was obtained for the pentapeptides Cys–Ile–His–Asn–Pro and Cys–Ile–Gln–Pro–Val while low response was achieved for the dipeptide. The comparative study, carried using a two-tailed T test, demonstrated that virtual screening was able to predict reliably the sensing ability of the pentapeptides. The dipeptide receptor exhibited 29% of virtual-experimental matching vs. 71% of glutathione and up to 93% for the pentapeptides. This virtual screening approach was proved to be a promising tool in predicting the behaviour of sensors array for gas detection.

A C-type lectin (AiCTL-3) from bay scallop Argopecten irradians with mannose/galactose binding ability to bind various bacteria

C-type lectins are a family of Ca2+-dependent carbohydrate-binding proteins playing crucial roles in innate immunity of vertebrates and invertebrates. In the present study, the cDNA of a C-type lectin with one carbohydrate-recognition domain (CRD) of 127 amino acids was cloned from bay scallop Argopecten irradians (designated AiCTL-3) by rapid amplification of cDNA end (RACE) techniques based on expressed sequence tag (EST) analysis. The mRNA transcripts of AiCTL-3 could be detected in all the tested tissues including hepatopancreas, gonad, adductor muscle, heart, hemocytes, mantle and gill, with the highest expression level in hepatopancreas. After the challenges with Vibrio anguillarum and Micrococcus luteus, the mRNA expression level of AiCTL-3 was obviously up-regulated and reached the maximum level at 9h (11.87fold, P<0.01, and 20.02-fold, P<0.05, respectively). The recombinant AiCTL-3 (designated as rAiCTL-3) could bind LPS, PGN, and glucan in vitro, but could not bind mannan. And it also bound Gram-positive bacteria Staphylococcus aureus as well as Gram-negative bacteria Escherichia coli and V. anguillarum. With a Ca2+ binding site 2 EPN (Glu–Pro–Asn) motif, rAiCTL-3 could bind both mannose and galactose which was quite different from those in vertebrate. Meanwhile, it could significantly enhance the phagocytosis of scallop hemocytes in vitro. The results clearly suggested that AiCTL-3 could serve not only as a PRR participated in the immune response against various PAMPs and bacteria in non-self recognition via mannose/galactose binding specificity but an opsonin playing an important part in clearance of invaders.

Calculation of the Three-Dimensional Structures of Two Antigenic Sequences, Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro and Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–Pro–Pro, of the Circumsporozoite Protein From a Malaria-Causing Plasmodium

Using the chain build-up procedure based on the program ECEPP, we have computed the lowest energy structures for two terminally blocked subsequences from the antigenic circumsporozoite protein of Plasmodium berghei, that is known to cause malaria in animals. The full antigenic sequence is an octapeptide proline-rich tandem repeat, (Pro-Pro-Pro-Pro-Asn-Pro-Asn-Asp)(2). We computed the structures for the first octapeptide plus one Pro from the second octapeptide, terminally blocked CH(3)CO-Pro-Pro-Pro-Pro-Asn-Pro-Asn-Asp-Pro-NHCH(3) as well as the first octpeptide with an additional three Pro residues from the adjoining unit, i.e., CH(3)CO-Pro-Pro-Pro-Pro-Asn-Pro-Asn-Asp-Pro-Pro-Pro-NHCH(3). We find that the first sequence adopts a number of different low energy structures, the most probable of which has a probability of occurrence of 56%. Addition of two more Pro residues results in the adoption a single, unique lowest energy structure that has a probability of occurrence of over 95% without solvation effects and 86% when solvation effects are included in the calculations. We predict that this structure may be the one recognized as a major antigenic determinant.

Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes

E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His-Pro-Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.

Bioorganic studies on the venom from duckbill platypus

Venomous mammals are rare, and only a few species in the orders Insectivora and Monotremata produce toxic venom. Among them, the duckbill platypus (Ornithorhynchus anatinus) is one of the two venomous Australian mammals. The adult male platypus carries a spur on each hind leg, which it uses to inject competitors with poison. However, the structure and function of the poison’s active compounds are still imcompletely characterized. We found that crude platypus venom produced potent Ca2+influx in human neuroblastoma IMR-32 cells. Guided by this assay, we identified 11 unique peptides, including peptide H–His–Asp–His–Pro–Asn–Pro–Arg–OH, which coincided with the N-terminal domain residues ofOrnithorhynchusvenom C-type natriuretic peptide (OvCNP). This heptapeptide induced a significant increase in [Ca2+]iin IMR-32 cells at 75 μM; had relatively specific affinities for glutamate, histamine, and GABAAreceptors; and facilitated neurogenic twitching in guinea pig ileum specimens at 30 μM. We also established that its proteinous venom fraction strongly hydrolyzed Pro–Phe–Arg–MCA and cleaved a human low-molecular-weight kininogen (LK), similar to porcine pancreas kallikrein. These results strongly indicated that platypus venom contains tissue kallikrein-like protease(s), and its proteolytic activity might synergistically contribute to toxicity through the specific cleavage of other venom constituents.

Combined use of LC–ESI-MS and antifungal tests for rapid identification of bioactive lipopeptides produced by Bacillus amyloliquefaciens CCMI 1051

The strain Bacillus amyloliquefaciens CCMI 1051 used in this study has been isolated in our laboratory from healthy Quercus suber in the south of Portugal and shows high levels of antagonistic properties against filamentous fungi that attack forest products industry due to the production of bioactive peptides.A combined use of LC–ESI-MS and antifungal tests allowed a rapid identification of lipopeptides as active compounds produced. Applying autobiographic methods it was possible to obtain active compounds. LC–ESI-MS, a powerful tool for rapid identification, indicates the presence of lipopeptides and MS2 electrospray ionization showed the partial sequence Tyr–Asn–Pro–Glu in the peptidic portion of some compounds produced. The association of mass spectrometry and chromatography, used in parallel with antifungal tests proved to be an efficient approach for the characterization of active lipopeptides without the need of previous total isolation. This methodology can be employed for screening and optimization the production of antifungal iturinic lipopeptides, showing a great potential for future application.

Recognition of Photoresponsive Polymer Targets by Protein Fused with cis-Form Azobenzene-binding Peptide

Abstract The Trp–His–Thr–Leu–Pro–Asn–Ala sequence, which preferentially recognizes cis-form azobenzene groups of synthetic polymers, was fused to the N-terminus of green fluorescent protein. Quartz crystal microbalance measurements were used to investigate its absorption behavior on the polymer film surfaces. The results indicated that the peptide succeeded in immobilizing the fused protein onto the film surfaces containing cis-form azobenzene groups.

Corrigendum to “A novel TRH analog, Glp–Asn–Pro–d-Tyr–d-TrpNH2, binds to [3H][3-Me-His2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2” [Neurosci. Lett. 431 (2007) 26–30

Corrigendum to “A novel TRH analog, Glp–Asn–Pro–d-Tyr–d-TrpNH2, binds to [3H][3-Me-His2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2” [Neurosci. Lett. 431 (2007) 26–30

A novel TRH analog, Glp–Asn–Pro– d-Tyr– d-TrpNH 2, binds to [ 3H][3-Me-His 2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2

Glp–Asn–Pro– d-Tyr– d-TrpNH 2 is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all- l counterpart (Glp–Asn–Pro–Tyr–TrpNH 2) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [ 3H][3-Me-His 2]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His 2]TRH potently displaced [ 3H][3-Me-His 2]TRH binding from all tissues/cells investigated. Glp–Asn–Pro– d-Tyr– d–TrpNH 2 in concentrations up to 10 −5 M did not displace [ 3H][3-Me-His 2]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all- l peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC 50 Glp–Asn–Pro– d-Tyr– d-TrpNH 2: 7.7 ± 0.2; pIC 50 Glp–Asn–Pro–Tyr–TrpNH 2: 6.6 ± 0.2), analogous to cortical membranes (pIC 50 Glp–Asn–Pro– d-Tyr– d-TrpNH 2: 7.8 ± 0.2; pIC 50 Glp–Asn–Pro–Tyr–TrpNH 2: 6.6 ± 0.2). Neither peptide, however, displaced [ 3H][3-Me-His 2]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp–Asn–Pro– d-Tyr– d-TrpNH 2 is not displacing [ 3H][3-Me-His 2]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.

Separation of low-molecular mass peptides by capillary electrophoresis with the use of alkylamines as dynamic coating agents at low pH

Modification of the silanophilic activity of the inner surface of the capillary wall was studied in a capillary electrophoretic system using alkylamines containing background electrolytes at acid pH. The effect of the following amine additives was investigated: (1) alkyl-α,ω-diamines (1,2-diaminoethane, 1,4-diaminobutane, 1,7-diaminoheptane, spermine), (2) polymeric amines (polyethylenimine, polybrene), (3) cationic amine surfactants (cetrimide, hexamethonium bromide). A seven membered test mixture of peptides (Gly–Pro–Ala, Pro–hPro, Gly–Pro–Arg, Gly–Pro–Gln, Lys–Pro–Gly, Asn–Pro–Gly, His–Pro–Gly) possessing one or more amino groups was used for selectivity evaluation. Under optimised concentration of the amine modifiers the selectivity was always improved (except for polybrene), particularly with the fast moving analytes. The best results were obtained with 1,2-diaminoethane and 1,7-diaminoheptane. On the other hand with slowly moving peaks the best separations were obtained with 1,7-diaminoheptane, hexadecyltrimethylammonium bromide and hexamethonium bromide, i.e. with modifiers possessing large aliphatic domains which are likely to be hydrophobically bonded with the separated solutes. The selectivity improvement with fast moving members of the test mixture can be ascribed to the decrease of the electroosmotic flow, while the improved separation with slowly moving peaks appears to reflect the altered interaction with the hydrophobized capillary wall. As expected the endoosmotic flow was in all cases decreased. The practical applicability of using amine based dynamic modifiers of the capillary wall was demonstrated on a natural peptide mixture (bacterial collagenase hydrolysate of collagen types I and III).

Single chain antibodies that recognize the N-glycosylation site

We aimed to identify antibodies that can recognize the Asn–Xaa–Ser/Thr(NXS/T) N-glycosylation site that guides oligosaccharyltransferase (OT) activity. We used synthetic Asn–Cys–Ser/Thr(NCS/T) tripeptides conjugated to bovine serum albumin to isolate single chain antibody fragments of a variable region (scFv) from the Griffin 1 phage antibody library. Although Ser and Thr have different side chains, the scFv proteins thus isolated bound to both NCS and NCT with K d values of the order of 10 −6 M and accepted the substitution of the Cys residue with various amino acids, including Ala, Gly, and Val. However, these proteins recognized neither Asn–Pro–Ser/Thr nor non-NXS/T tripeptides. The scFv proteins recognized NCS/T and N-glycosylation site of mutant yeast protein disulfide isomerase when they were in their native but not denatured state. These results indicate that antibody recognition of the NXS/T motif is conformation dependent and suggest that NXS/T spontaneously adopts a specific conformation that is necessary for antibody recognition. These features are likely to correlate with the known binding specificity of OT.

Encoding Selectivity of a Transmembrane Channel.

Membrane channel proteins of the aquaporin family are integral plasma membrane proteins that are highly selective for permeation of specific small molecules, with absolute exclusion of ions including protons or OHanions and charged solutes, and without dissipating the electrochemical potential across the cell membrane. There are 11 human family members. The family includes channels that conduct water, called aquaporins, and channels that conduct glycerol or other linear polyalcohols (aquaglyceroporins), and ion conducting channels. The E. coli glycerol facilitator (GlpF) was cloned, expressed, purified, and crystallized with its primary permeant substrate glycerol. The crystal structure of GlpF, determined by multiple isomorphous replacement at 2.2 A resolution illustrate the basis for its selectivity. Glycerol molecules G1, G2, G3 line up in an amphipathic channel in single file. In the narrow selectivity filter of the channel, the glycerol alkyl backbone is wedged against a hydrophobic corner and successive OH groups form hydrogen bonds with a pair of acceptor, and donor atoms. These hydrogen bonds serve to orient the CHXOH groups within the narrower regions of the channel, and explain the stereo, and enantio selectivity measured for this channel. Two conserved -Asn Pro Alamotifs meet in the center of the membrane and form a conserved interface between two gene-duplicated segments that each encode three and one half membrane spanning helices around the channel. This structure elucidates the mechanism of selective permeability for linear carbohydrates and suggests how ions and water are excluded. A functional assay was developed, and shows that the channel is both stereoand enantio-selective. The conservation of sequence shows how the water channels of the same family conduct water and exclude glycerol.

Differentiated reactivity of whole blood neonatal platelets to various agonists

Neonatal platelets have been occasionally reported to show a reduced response to various agonists. The molecular mechanism(s) of such a depressed reactivity remains unclear. To further address this problem we studied neonatal platelet activation with thrombin, TRAP (thrombin receptor activating peptide, Ser–Phe– Leu–Leu–Arg–Asn–Pro–Asn–Asp–LysTyrGlu and ADP in 42 healthy 1–2 day old neonates using a whole peripheral blood flow cytometry. The neonates did not show an increased fraction of P-selectinpositive circulating platelets, whereas the expression of GPIb (glycoprotein Ib) in resting neonatal platelets was significantly lower compared to adults. Neonatal platelets were significantly less reactive than adult platelets to thrombin and TRAP, especially at lower agonist concentrations, but not to ADP or when incubated for 1 h at room temperature. Activation of neonatal platelets with agonists resulted in a marked alterations in the expression of P-selectin, whereas the internalization of GPIb was not affected. The reduced neonatal platelet sensitivity to thrombin and TRAP was accompanied by significantly reduced ATIII (antithrombin III) and increased prothrombin fragment F 1+2 in neonatal plasma. We conclude that various receptor systems potentially able to bind thrombin are relatively insensitive in neonatal platelets. The novelty of our work is that neonatal platelet hyposensitivity is not a generalized phenomenon, but concerns only selected agonists and selected receptor systems.