Effects of the new antiarrhythmic peptide ZP123 on epicardial activation and repolarization pattern

Antiarrhythmic peptides such as AAP10 (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH(2)) have antiarrhythmic properties related to their stimulatory effect on gap junctional coupling. However, most of these peptides are not stable in enzymatic environment which limits studies with these compounds in vivo. ZP123 is a new antiarrhythmic peptide constructed using a retro-all-D-amino acid design of the AAP10 template (Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH(2)). The aim of this study was to compare the effects of AAP10 and ZP123 on epicardial activation and repolarization patterns in isolated perfused rabbit hearts. In addition, we tested the effect of these compounds on PKC activation in cultured HeLa-Cx43 cells. Rabbit hearts were perfused according to the Langendorff technique with Tyrode solution at constant pressure (70 cm H(2)O). After 45 min equilibration, either AAP10 (n = 7) or ZP123 (n = 7) was infused intracoronarily in concentrations of 0.1, 1, 10, 100, and 1000 nM (15 min for each concentration) in the presence of 0.05% bovine serum albumine. 256 AgCl electrodes were attached to the hearts surface and connected to the inputs of a 256 channel mapping system in a unipolar circuit (4 kHz/channel, 0.04 mV vertical resolution, 1 mm spatial resolution). For each electrode the activation and repolarization timepoint were determined. We found that both peptides significantly reduced epicardial dispersion by a maximum of about 20% thereby enhancing the homogeneity of epicardial action potential duration, while the action potential duration itself was not affected. The beat-to-beat variability of the epicardial activation pattern was stabilized by both peptides as compared to an untreated time-control series. Other parameters such as LVP, CF, heart rate, or total activation time were not effected by either of the peptides. In a second protocol, rectangular pulses were delivered to the back wall and the propagation velocity was determined longitudinal and transversal to the fiber axis. We found an increase in both longitudinal and transversal conduction velocity. Using a commercial PKC assay on HeLa-Cx43 cells we found that 50 nM AAP10 and 50 nM ZP123 increased activity by 99 +/- 6% and 146 +/- 54%, respectively. The PKC activation induced by either of these compounds was completely blocked using the selective PKCalpha inhibitor GCP54345. We conclude that AAP10 and ZP123 have similar effects in vitro, but the superior enzymatic stability of ZP123 makes this compound the preferred substance for in vivo studies of antiarrhythmic peptides.     

Protein kinase Calpha mediates the effect of antiarrhythmic peptide on gap junction conductance

We investigated the effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH2, 50 nM) on pairs of adult guinea pig cardiomyocytes and on pairs of HeLa-cells transfected with rat connexin43 (Cx43). Using double cell voltage clamp technique in cardiomyocytes under control conditions, gap junction conductance (Gj) steadily decreased (by -0.3 to -0.4 nS/min). In contrast, 50 nM AAP10 significantly enhanced Gj (by +0.22 to +0.29 nS/min). This effect of AAP10 could be significantly antagonized by bisindolylmaleimide I (BIM), and by the protein kinase C (PKC) subtype-specific inhibitors HBDDE (PKCgamma and -alpha) and CGP 54345 (PKCalpha). In HeLa-Cx43 cells we found similar electrophysiological effects of AAP10. For further analysis, we incubated HeLa-Cx43 cells with [32P]orthophosphate (0.05 mCi/ml) for 4 h at 37 degrees C followed by addition of 50 nM AAP10 for 15 min. We found that incorporation of 32P into Cx43 was significantly enhanced in the presence of AAP10, which was completely inhibited in presence of BIM. PKC enzyme-linked immunosorbent assay (ELISA) revealed significant activation of PKC by AAP10 in HeLa-Cx43 cells, which could be inhibited by HBDDE and CGP 54345. Finally, a binding study using [14C]-AAP10 as radioligand was performed. We found a saturable binding of [14C]-AAP10 with a KD of 0.88 nM to cardiac membrane preparations. For assessment of the antiarrhythmic activity in anesthetized rats, we infused aconitine until the occurrence of ventricular fibrillation (VF). The aconitine dose required for initiation of VF was significantly enhanced in the presence of AAP10. In conclusion; AAP10 increases Gj in both adult cardiomyocytes and transfected HeLa-Cx43 cells. AAP10 leads to enhanced phosphorylation of Cx43 via activation of PKCalpha. A membrane receptor exists for antiarrhythmic peptides.     

Effects of the New Antiarrhythmic Peptide ZP123 on Epicardial Activation and Repolarization Pattern

Antiarrhythmic peptides such as AAP10 (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH₂) have antiarrhythmic properties related to their stimulatory effect on gap junctional coupling. However, most of these peptides are not stable in enzymatic environment which limits studies with these compounds in vivo. ZP123 is a new antiarrhythmic peptide constructed using a retro-all-D-amino acid design of the AAP10 template (Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH₂). The aim of this study was to compare the effects of AAP10 and ZP123 on epicardial activation and repolarization patterns in isolated perfused rabbit hearts. In addition, we tested the effect of these compounds on PKC activation in cultured HeLa-Cx43 cells. Rabbit hearts were perfused according to the Langendorff technique with Tyrode solution at constant pressure (70 cm H₂O). After 45 min equilibration, either AAP10 (n = 7) or ZP123 (n = 7) was infused intracoronarily in concentrations of 0.1, 1, 10, 100, and 1000 nM (15 min for each concentration) in the presence of 0.05% bovine serum albumine. 256 AgCl electrodes were attached to the hearts surface and connected to the inputs of a 256 channel mapping system in a unipolar circuit (4 kHz/channel, 0.04 mV vertical resolution, 1 mm spatial resolution). For each electrode the activation and repolarization timepoint were determined. We found that both peptides significantly reduced epicardial dispersion by a maximum of about 20% thereby enhancing the homogeneity of epicardial action potential duration, while the action potential duration itself was not affected. The beat-to-beat variability of the epicardial activation pattern was stabilized by both peptides as compared to an untreated time-control series. Other parameters such as LVP, CF, heart rate, or total activation time were not effected by either of the peptides. In a second protocol, rectangular pulses were delivered to the back wall and the propagation velocity was determined longitudinal and transversal to the fiber axis. We found an increase in both longitudinal and transversal conduction velocity. Using a commercial PKC assay on HeLa-Cx43 cells we found that 50 nM AAP10 and 50 nM ZP123 increased activity by 99 ± 6% and 146 ± 54%, respectively. The PKC activation induced by either of these compounds was completely blocked using the selective PKCα inhibitor GCP54345. We conclude that AAP10 and ZP123 have similar effects in vitro, but the superior enzymatic stability of ZP123 makes this compound the preferred substance for in vivostudies of antiarrhythmic peptides.     

Protein Kinase Cα Mediates the Effect of Antiarrhythmic Peptide on Gap Junction Conductance

We investigated the effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH₂, 50 nM) on pairs of adult guinea pig cardiomyocytes and on pairs of HeLa-cells transfected with rat connexin43 (Cx43). Using double cell voltage clamp technique in cardiomyocytes under control conditions, gap junction conductance (G_j) steadily decreased (by -0.3 to -0.4 nS/min). In contrast, 50 nM AAP10 significantly enhanced G_j (by +0.22 to +0.29 nS/min). This effect of AAP10 could be significantly antagonized by bisindolylmaleimide I (BIM), and by the protein kinase C (PKC) subtype-specific inhibitors HBDDE (PKCγ and -α) and CGP 54345 (PKCα). In HeLa-Cx43 cells we found similar electrophysiological effects of AAP 10. For further analysis, we incubated HeLa-Cx43 cells with [³²P]orthophosphate (0.05 mCi/ml) for 4 h at 37°C followed by addition of 50 nM AAP10 for 15 min. We found that incorporation of ³²P into Cx43 was significantly enhanced in the presence of AAP 10, which was completely inhibited in presence of BIM. PKC enzyme-linked immunosorbent assay (ELISA) revealed significant activation of PKC by AAP10 in HeLa-Cx43 cells, which could be inhibited by HBDDE and CGP 54345. Finally, a binding study using [¹⁴C]-AAP10 as radioligand was performed. We found a saturable binding of [¹⁴C]-AAP10 with a K₀ of 0.88 nM to cardiac membrane preparations. For assessment of the antiarrhythmic activity in anesthetized rats, we infused aconitine until the occurrence of ventricular fibrillation (VF). The aconitine dose required for initiation of VF was significantly enhanced in the presence of AAP 10. In conclusion; AAP 10 increases G_j in both adult cardiomyocytes and transfected HeLa-Cx43 cells. AAP 10 leads to enhanced phosphorylation of Cx43 via activation of PKCα. A membrane receptor exists for antiarrhythmic peptides.     

Theoretical studies of the structure and molecular dynamics of a peptide crystal

Energy minimizations and molecular dynamics simulations have been performed on the cyclic peptide cyclo-(Ala-Pro-D-Phe)2 in both the isolated and crystal states. The results of these calculations have been analyzed, both to investigate our ability to reproduce experimental data (structure and vibrational and NMR spectra) and to investigate the effects of environment on the energy, structure, and dynamics of peptides. Comparison of the minimized and time-averaged crystal systems with the experimental peptide structure shows that the calculations have closely reproduced the experimental structure. Molecular dynamics of the isolated molecule has led to a new conformation, which is approximately equal to 8.5 kcal/mol more stable than the conformation that exists in the crystal, the latter conformation being stabilized by intermolecular (packing) forces. This illustrates the considerable effect that environment can have on the conformation of peptides. The crystal environment has also been shown to significantly reduce the dynamic conformational fluctuations seen for the isolated molecule. The behavior of the peptide during the isolated simulation also supports the experimental NMR observation of a symmetric structure that differs from the asymmetric, instantaneous structures which characterize the molecule during the dynamics. Calculations of vibrational frequencies of the peptide in the crystal and isolated states show the expected shifts in bond-stretching frequencies due to intermolecular interactions. Finally, we have calculated NMR coupling constants from the dynamics simulation of the isolated peptide and have compared these with the experimental values. This has led to a possible reinterpretation of the experimental data.     

The 1.20 A resolution crystal structure of the aminopeptidase from Aeromonas proteolytica complexed with tris: a tale of buffer inhibition

The aminopeptidase from Aeromonas proteolytica (AAP) is a bridged bimetallic enzyme that removes the N-terminal amino acid from a peptide chain. To fully understand the metal roles in the reaction pathway of AAP we have solved the 1.20 A resolution crystal structure of native AAP (PDB ID = 1LOK). The high-quality electron density maps showed a single Tris molecule chelated to the active site Zn(2+), alternate side chain conformations for some side chains, a sodium ion that mediates a crystal contact, a surface thiocyanate ion, and several potential hydrogen atoms. In addition, the high precision of the atomic positions has led to insight into the protonation states of some of the active site amino acid side chains.     

Spatial structure determination of antiarrhythmic peptide using nuclear magnetic resonance spectroscopy

The peptide AAP10 was synthesized according to the Merrifield technique following the Fmoc-strategy and its spatial structure in aqueous solution studied with NMR principles. It is known from previous studies that the peptide has antiarrhythmic activity and inhibits cardiac ischemia induced alterations of the activation pattern, decreases the activation–recovery interval (ARI) dispersion and improves cellular coupling via enhancement of gap junction conductance (2, 2, 3, 4). The peptide was synthesized as a peptide amide. Two different semi cyclic conformations were characterized.     

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides.

Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).     

Function of the signal peptide and N- and C-terminal propeptides in the leucine aminopeptidase from Aeromonas proteolytica

The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP's native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type AAP to be proposed. The role of the A. proteolytica signal peptide in protein secretion as well as comparison to other known signal peptides reveals a close resemblance of the A. proteolytica signal peptide to the outer membrane protein (OmpA) signal peptide. Over-expression of the full 54 kDa AAP enzyme provides an enzyme that is significantly less active, due to a cooperative inhibitory interaction between both propeptides. Over-expression of AAP lacking its C-terminal propeptide provided an enzyme with an identical kcat value to wild-type AAP but exhibited a larger Km value, suggesting competitive inhibition of AAP by the N-terminal propeptide (Ki approximately 0.13 nM). The recombinant 32 kDa form of AAP was characterized by kinetic and spectroscopic methods and was shown to be identical to mature, wild-type AAP. Therefore, the ease of purification and processing of rAAP along with the fact that large quantities can be obtained now allow new detailed mechanistic studies to be performed on AAP through site-directed mutagenesis.     

Spectroscopic and X-ray crystallographic characterization of bestatin bound to the aminopeptidase from Aeromonas (Vibrio) proteolytica

Binding of the competitive, slow-binding inhibitor bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoy]-leucine) to the aminopeptidase from Aeromonas proteolytica (AAP) was examined by both spectroscopic and crystallographic methods. Electronic absorption spectra of the catalytically competent [Co_(AAP)], [CoCo(AAP)], and [ZnCo(AAP)] enzymes recorded in the presence of bestatin revealed that both of the divalent metal ions in AAP are involved in binding bestatin. The electron paramagnetic resonance (EPR) spectrum of the [CoCo(AAP)]-bestatin complex exhibited no observable perpendicular- or parallel-mode signal. These data indicate that the two Co(II) ions in AAP are antiferromagnetically coupled yielding an S = 0 ground state and suggest that a single oxygen atom bridges between the two divalent metal ions. The EPR data obtained for [CoZn(AAP)] and [ZnCo(AAP)] confirm that bestatin interacts with both metal ions. The X-ray crystal structure of the [ZnZn(AAP)]-bestatin complex was solved to 2.0 A resolution. Both side chains of bestatin occupy a well-defined hydrophobic pocket that is adjacent to the dinuclear Zn(II) active site. The amino acid residues ligated to the dizinc(II) cluster in AAP are identical to those in the native structure with only minor perturbations in bond length. The alkoxide oxygen of bestatin bridges between the two Zn(II) ions in the active site, displacing the bridging water molecule observed in the native [ZnZn(AAP)] structure. The M-M distances observed in the AAP-bestatin complex and native AAP are identical (3.5 A) with alkoxide oxygen atom distances of 2.1 and 1.9 A from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 A. In addition, the NH(2) group of bestatin, which mimics the N-terminal amine group of an incoming peptide, binds to Zn2 with a bond distance of 2.3 A. A combination of the spectroscopic and X-ray crystallographic data presented herein with the previously reported mechanistic data for AAP has provided additional insight into the substrate-binding step of peptide hydrolysis as well as insight into important small molecule features for inhibitor design.     

Nonenzymatic lipid mediators,neuroprostanes, exert the antiarrhythmic properties of docosahexaenoic acid

Neuroprostanes are lipid mediators produced by nonenzymatic free radical peroxidation of docosahexaenoic acid (DHA). DHA is associated with a lower atherosclerosis risk, suggesting a beneficial role in cardiovascular diseases. The aim of this study was to investigate the influence of DHA peroxidation on its potentially antiarrhythmic properties (AAP) in isolated ventricular cardiomyocytes and in vivo in post-myocardial infarcted mice. Calcium imaging and biochemical experiments indicate that cardiac arrhythmias induced by isoproterenol are associated with Ca²⁺ leak from the sarcoplasmic reticulum after oxidation and phosphorylation of the type 2 ryanodine receptor (RyR2) leading to dissociation of the FKBP12.6/RyR2 complex. Both oxidized DHA and 4(RS)-4-F_4t-NeuroP prevented cellular arrhythmias and posttranslational modifications of the RyR2 leading to a stabilized FKBP12.6/RyR2 complex. DHA per se did not have AAP. The AAP of 4(RS)-4-F_4t-NeuroP was also observed in vivo. In this study, we challenged the paradigm that spontaneously formed oxygenated metabolites of lipids are undesirable as they are unconditionally toxic. This study reveals that the lipid mediator 4(RS)-4-F_4t-neuroprostane derived from nonenzymatic peroxidation of docosahexaenoic acid can counteract such deleterious effects through cardiac antiarrhythmic properties. Our findings demonstrate 4(RS)-4-F_4t-NeuroP as a mediator of the cardioprotective AAP of DHA. This discovery opens new perspectives for products of nonenzymatic oxidized ω3 polyunsaturated fatty acids as potent mediators in diseases that involve ryanodine complex destabilization such as ischemic events.     

Primary structure of the OSCP protein that confers sensitivity to oligomycin on the mitochondrial H+-ATPase complex. I. Tryptic and cyanogen bromide peptides

Trypsin and cyanogen bromide were used for cleavage of the OSCP preparations. The peptide mixtures thus formed were separated into individual components by a combination of various chromatographic procedures: gel filtration, ion exchange and paper chromatography, as well as reversed-phase HPLC. As a result, 31 tryptic peptides and 9 out of 10 possible cyanogen bromide peptides were isolated. Determination of the amino acid sequences of these peptide allowed the alignment of cyanogen bromide fragments in the polypeptide chain that shed light on the "architecture" of the protein molecule as a whole. It also afforded the overlappings for tryptic peptides, 16 in the N-terminal and 8 in the C-terminal portions of the molecule.     

Modeling an active conformation for linear peptides and design of a competitive inhibitor for HMG-CoA reductase

This study presents an approach that can be used to search for lead peptide candidates, including unconstrained structures in a recognized sequence. This approach was performed using the design of a competitive inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). In a previous design for constrained peptides, a head-to-tail cyclic structure of peptide was used as a model of linear analog in searches for lead peptides with a structure close to an active conformation. Analysis of the conformational space occupied by the peptides suggests that an analogical approach can be applied for finding a lead peptide with an unconstrained structure in a recognized sequence via modeling a cycle using fixed residues of the peptide backbone. Using the space obtained by an analysis of the bioactive conformations of statins, eight cyclic peptides were selected for a peptide library based on the YVAE sequence as a recognized motif. For each cycle, the four models were assessed according to the design criterion ("V" parameter) applied for constrained peptides. Three cyclic peptides (FGYVAE, FPYVAE, and FFYVAE) were selected as lead cycles from the library. The linear FGYVAE peptide (IC(50) = 0.4 microM) showed a 1200-fold increase the inhibitory activity compared to the first isolated LPYP peptide (IC(50) = 484 microM) from soybean. Experimental analysis of the modeled peptide structures confirms the appropriateness of the proposed approach for the modeling of active conformations of peptides.     

Changing ABRA protein peptide to fit into the HLA-DRbeta1*0301 molecule renders it protection-inducing

The Plasmodium falciparum acidic-basic repeat antigen represents a potential malarial vaccine candidate. One of this protein's high activity binding peptides, named 2150 ((161)KMNMLKENVDYIQKNQNLFK(180)), is conserved, non-immunogenic, and non-protection-inducing. Analogue peptides whose critical binding residues (in bold) were replaced by amino-acids having similar mass but different charge were synthesized and tested to try to modify such immunological properties. These analogues' HLA-DRbeta1* molecule binding ability were also studied in an attempt to explain their biological mechanisms and correlate binding capacity and immunological function with their three-dimensional structure determined by (1)H NMR. A 3(10) distorted helical structure was identified in protective and immunogenic peptide 24922 whilst alpha-helical structure was found for non-immunogenic, non-protective peptides having differences in alpha-helical position. The changes performed on immunogenic, protection-inducing peptide 24922 allowed it to bind specifically to the HLA-DRbeta1*0301 molecule, suggesting that these changes may lead to better interaction with the MHC Class II-peptide-TCR complex rendering it immunogenic and protective, thus suggesting a new way of developing multi-component, sub-unit-based anti-malarial vaccines.     

In silico study of M18 aspartyl amino peptidase (M18AAP) of Plasmodium vivax as an antimalarial drug target

Plasmodium vivax (Pv) is the second most malaria causing pathogen among Plasmodium species. M18 aspartic aminopeptidase (M18AAP) protein is a single gene copy present in Plasmodium. This protein is functional at the terminal stage of hemoglobin degradation of host and completes the hydrolysis process which makes it an important target for new chemotherapeutics. No experimental and structural study on M18AAP protein of P. vivax is reported till today. This paper advocates the application of multiple computational approaches like protein model prediction, ligand-based 3D QSAR study, pharmacophore, structure-based virtual screening and molecular docking simulation for identification of potent lead molecules against the enzyme. The 3D QSAR model was developed using known bioactive compounds against the PvM18AAP protein which statistically signify the k-NN model with q^2 = 0.7654. The study reports a lead molecule from ligand-centric approach with good binding affinity and possessing lowest docking score. The findings will be helpful for in-vivo and in-vitro validations and development of potent anti-malarial molecules against the drug resistant strains of malaria parasite.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

Phage matrix for isolation of glioma cell membrane proteins

Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.     

Abundance, depth distribution, and composition of aerobic bacteriochlorophyll a-producing bacteria in four basins of the central Baltic Sea

The abundance, vertical distribution, and diversity of aerobic anoxygenic phototrophic bacteria (AAP) were studied at four basins of the Baltic Sea. AAP were enumerated by infrared epifluorescence microscopy, and their diversity was analyzed by using pufM gene clone libraries. In addition, numbers of CFU containing the pufM gene were determined, and representative strains were isolated. Both approaches indicated that AAP reached maximal abundance in the euphotic zone. Maximal AAP abundance was 2.5 x 10(5) cells ml(-1) (11% of total prokaryotes) or 1.0 x 10(3) CFU ml(-1) (9 to 10% of total CFU). Environmental pufM clone sequences were grouped into 11 operational taxonomic units phylogenetically related to cultivated members of the Alpha-, Beta-, and Gammaproteobacteria. In spite of varying pufM compositions, five clones were present in all libraries. Of these, Jannaschia-related clones were always found in relative abundances representing 25 to 30% of the total AAP clones. The abundances of the other clones varied. Clones potentially affiliated with typical freshwater Betaproteobacteria sequences were present at three Baltic Sea stations, whereas clones grouping with Loktanella represented 40% of the total cell numbers in the Gotland Basin. For three alphaproteobacterial clones, probable pufM phylogenetic relationships were supported by 16S rRNA gene analyses of Baltic AAP isolates, which showed nearly identical pufM sequences. Our data indicate that the studied AAP assemblages represented a mixture of marine and freshwater taxa, thus characterizing the Baltic Sea as a "melting pot" of abundant, polyphyletic aerobic photoheterotrophic bacteria.     

Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study

Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20, which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.