The use of valved conduits in pediatric cardiac surgery

A library system was developed for the discovery of bioactive peptides. Library synthesis and peptide sequencing was performed on a solid support while the screening for bioactivity was done with peptides in solution. The peptides were synthesized by split and mix, one-bead-one-peptide library synthesis, using a Tentagel S-NH2 solid support with a loading of approximately 100 pmol/bead. The major part of the peptide was connected to the support by a single acid-labile linker and a minor part of the peptide was acid-stabile attached to the polymer. The percentage of acid-stabile attached peptides could easily be controlled during modification of the amino functionalities of the resin at the start of the process. The cleavage rate of the acid-labile attached peptide from the resin depends on the composition of the cleavage mixture. When cleavage conditions were carefully controlled, a three-step partial cleavage protocol allowed for convergent bioactivity screening on peptide libraries using only one type of acid-labile linker. The partial cleavage and convergent screening procedure was repeated three times, after which the bead containing the bioactive peptide was sequenced. As such a bead still contained acid-stabile attached peptide, the Edman sequencing was straightforward and repetitive yields were excellent because the immobilized peptide was not washed out.     

Structure/antigenicity relationship of cyclic and linear peptides mimicking the V3 loop of HIV2 envelope glycoprotein

We report the structure and antigenicity of the third variable region (V3) of the HIV2 envelope glycoprotein by the use of linear and cyclic peptides. To this end, a peptide mimicking this region was synthesized and purified, both as an iodoacetamidated linear peptide and a disulphide-bridged cyclic peptide. The cross-reactivity of three monoclonal antibodies (mAbs) produced against the envelope glycoprotein gp140 with the linear and cyclic peptides was tested with ELISA. The results showed that the cyclic peptide is a better ligand for the 3 mAbs 125-F, 125-J and 125-K. The avidity of the mAb/peptide interaction was further analysed by determining the concentration of linear or cyclic peptide leading to 50% inhibition of mAb-peptide complex formation (K0.5). The K0.5 value of mAb 125-F, which displayed the best reactivity with gp140, was estimated to be 5 times higher for the linear (K0.5 = 1.5 x 10(-6) M) than for the cyclic peptide (K0.5 = 3 x 10(-7) M). This indicates a higher affinity of mAb 125-F for the cyclic peptide. mAb 125-J, which exhibited a lower avidity for the gp140 compared to mAb 125-F, had a similar affinity for the cyclic and the linear peptides (K0.5 = 3 x 10(-7) M). mAb 125-K had the lowest reactivity with gp140 and its binding to adsorbed peptide could not be inhibited by the soluble linear or cyclic peptide used up to 10(-5) M. These results suggest that cyclic peptides may have a higher propensity for adopting a native-like structure for the peptide/antibody interaction. Nuclear magnetic resonance experiments at 25 degrees C in phosphate buffer pH 5.4, however, showed that neither peptide displayed a well-defined structure.     

Localization of human transcription factor TEF-4 and TEF-5 (TEAD2, TEAD3) genes to chromosomes 19q13.3 and 6p21.2 using fluorescence in situ hybridization and radiation hybrid analysis

The tetrapeptide Ala-lle-Gly-Met bound to a Wang resin via the methionine residue was studied by NMR under MAS conditions and compared to the same peptide in solution. The bound peptide exhibits average linewidths superior to those observed for the peptide in solution. The origin of the residual NMR linewidth observed for the bound form was investigated. The dynamics of the peptide is shown to be only marginally responsible for the increased linewidth; the major cause of the line broadening appears to be nonaveraged magnetic susceptibility differences.     

Lung carcinoma imaging using a synthetic laminin derivative radioiodinated peptide YIGSR

The synthetic laminin pentapeptide tyrosyl-isoleucyl-glycyl-seryl-arginine (YIGSR) binds to a metastasis associated high-affinity laminin receptor. The aim of this study was to investigate if the radiolabeled peptide can be considered as a basis for a potential tumor-imaging radiopharmaceutical. METHODS: Iodine-131-labeled YIGSR was injected in mice inoculated with Lewis Lung carcinoma, as well as in normal controls. The experimental animals were imaged on a gamma camera 10 hr after peptide administration. The same peptide was also labeled with 125I and administered to tumor-bearing and normal mice. At various time-points after peptide administration, the experimental animals were killed, and the radioactivity in the tumor, lung, liver and spleen was measured. Microscopic autoradiography was performed in histological sections of the same tissues. RESULTS: The tumor and the spleen of tumor-bearing animals were imaged on a gamma camera. No significant blood-pool background was detected. No other organ except urinary bladder and thyroid was imaged in normal animals. The peptide was retained on tumor and spleen of tumor-bearing animals. Twenty-four hours after peptide administration, the tumor, lung, liver and spleen of animals with tumors contained significantly more radioactivity than the same tissues of equally treated normal controls. The radiolabeled peptide YIGSR was detected by microscopic autoradiography on the surface of certain tumor cells, but not on the surface of any normal cell. CONCLUSION: Although extensive research is still required, the peptide YIGSR can be considered as a basis for the development of a receptor specific radiopharmaceutical useful for the in vivo estimation of the metastatic potential of tumors. This radiopharmaceutical may be helpful in staging and prognostic-related decisions on cancer treatment.     

Calcitonin gene-related peptide promotes differentiation, but not survival, of rat mesencephalic dopaminergic neurons in vitro

The aim of this study was to investigate putative effects of calcitonin gene-related peptide on developing dopaminergic neurons in the ventral mesencephalon. To determine a time-point for a physiological role of calcitonin gene-related peptide in the development of this system, we first investigated calcitonin gene-related peptide messenger RNA expression in the ventral mesencephalon of Wistar rats at embryonic days (E) 11-19. Calcitonin gene-related peptide messenger RNA was not detectable at E11, i.e. prior to the appearance of dopaminergic neurons in this area. From E14 to E19, calcitonin gene-related peptide messenger RNA was expressed in increasing amounts. We therefore investigated the effects of calcitonin gene-related peptide on serum-free cell cultures established from the E14 midbrain floor. Addition of calcitonin gene-related peptide (200 ng/ml) every other day significantly increased neuronal differentiation, including longer tyrosine hydroxylase-positive neurites, enhanced immunoreactivity for growth-associated protein-43 and increased dopaminergic uptake per neuron. These effects were maximal after seven to eight days. Calcitonin gene-related peptide acted synergistically with fibroblast growth factor-2 on these parameters. In contrast to fibroblast growth factor-2, however, calcitonin gene-related peptide did not promote survival of tyrosine hydroxylase-immunoreactive neurons. Lack of calcitonin gene-related peptide expression in the mesencephalon at E11 was paralleled by a lack of effect of calcitonin gene-related peptide on early presumptive dopaminergic neurons in terms of eliciting this phenotype. Our data suggest that calcitonin gene-related peptide may act physiologically as a differentiation-promoting factor for phenotypically defined dopaminergic neurons during a time period when dopaminergic neurons assemble in the ventral mesencephalon and grow axons towards their targets.     

Increased cardiac workload by adrenoceptor agonists for the estimation of potential antiischemic activity in a conscious rabbit model

A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against alpha-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with alpha-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions. The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of alpha-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions. The inhibition constant for alpha-chymotrypsin was estimated to be 10(-6) M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified alpha-chymotrypsin than for natural alpha-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively prevented from binding to alpha-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe- Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of alpha-chymotrypsin and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature.     

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.     

Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland

CART peptide specific polyclonal antisera were raised in rabbits. The antisera were raised to CART peptide fragments that span most of the predicted CART protein. The specificity of each antisera was demonstrated by blockade of immunostaining by the immunizing peptide but not by the other CART peptide fragments. In the hypothalamus and pituitary of colchicine and noncolchicine treated rats, immunostaining was observed in cell bodies, fibers and varicosities. Clusters of cells were also stained in the adrenal medulla. It is noteworthy that cellular immunostaining was only found in areas previously shown to express CART mRNA. These findings indicate the presence of CART peptide(s) in the hypothalamus, pituitary, and adrenal gland. Furthermore, we also present evidence for the possible processing of the CART pro-peptide into smaller peptide fragments. These neuroanatomical findings suggest a role of CART peptides in hypothalamic, pituitary and adrenal function.     

An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae

Competence for genetic transformation in Streptococcus pneumoniae has been known for three decades to arise in growing cultures at a critical cell density, in response to a secreted protease-sensitive signal. We show that strain CP1200 produces a 17-residue peptide that induces cells of the species to develop competence. The sequence of the peptide was found to be H-Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg- Lys-Lys-OH. A synthetic peptide of the same sequence was shown to be biologically active in small quantities and to extend the range of conditions suitable for development of competence. Cognate codons in the pneumococcal chromosome indicate that the peptide is made ribosomally. As the gene encodes a prepeptide containing the Gly-Gly consensus processing site found in peptide bacteriocins, the peptide is likely to be exported by a specialized ATP-binding cassette transport protein as is characteristic of these bacteriocins. The hypothesis is presented that this transport protein is encoded by comA, previously shown to be required for elaboration of the pneumococcal competence activator.     

Increased immunoexpression of atrial natriuretic peptide in the heart conduction system of the rat after cardiac sympathectomy

The present investigation was designed to elucidate which role the sympathetic nerves play in the immunoexpression of atrial natriuretic peptide in the cardiac conduction system of the rat. In order to destroy the cardiac sympathetic nerve terminals, both surgical and chemical sympathectomy were performed. By use of immunohistochemical and radioimmunoassay techniques, the immunoreactivity and level of atrial natriuretic peptide in the conduction system and in the cardiac myocardium were determined. In contrast to the low degree of immunoreaction for atrial natriuretic peptide seen in control rats, the sympathectomized rats exhibited pronounced immunoreactivity for atrial natriuretic peptide in the atrioventricular bundle and bundle branches, which normally have high numbers of sympathetic nerve fibres. On the other hand, in the peripheral parts of the conduction system, where there are ordinarily few sympathetic nerve fibres, the degree of immunoreaction was unchanged. The quantitative measurements also showed that the entire ventricles, including the conduction system, contained increased levels of atrial natriuretic peptide in the treated hearts. The present study shows that destruction of the sympathetic nervous system leads to an increased level of atrial natriuretic peptide in the Purkinje fibres of bundle branches, which thus seem to have a dormant capacity for synthesis of this peptide. The results provide new evidence about the change in atrial natriuretic peptide levels that occurs when sympathetic innervation is altered.     

NMR studies on the conformation of the CD4 36-59 peptide bound to HIV-1 gp120

A peptide containing residues 36-59 of the human CD4 receptor includes most of the residues thought to be involved in binding the HIV surface glycoprotein, gp120. This peptide was synthesized and inhibited the binding of gp120 to soluble CD4. NMR relaxation experiments indicated that the peptide was in fast exchange between the free and gp120-bound states. Transferred NOESY NMR showed a number of long-range NOEs, from the gp120-bound state, between residues 38, 40, 45, 48, and 49 of the peptide. NMR evidence also suggested that the Phe43 in the peptide, which corresponds to a critical residue in CD4 for the binding of gp120, makes intimate contact with gp120. The Tr-NOESY cross-peak intensities provided proton-proton distance constraints on the conformation of the gp120-bound peptide. The distance constraints were used in simulated annealing, and a set of 20 very similar structures was obtained for the central region of the gp120-bound peptide. Residues 42-49 of the peptide formed a loop with the side chain of Phe43 pointing away from the rest of the peptide. This Phe43 ring points away from the protein surface in two structures of the amino-terminal domain of CD4 found by X-ray crystallography. Differences in the conformation of CD4 in the two crystal forms suggest that the 36-59 region might be flexible. The NMR data on the 36-59 CD4 peptide predicts a gp120-bound conformation different from either of the CD4 crystal forms in the absence of gp120.     

Postdural puncture headache and back pain after spinal anesthesia with 27-gauge Quincke and 26-gauge Atraucan needles

A 17-residue peptide containing the caulimovirus-related "zinc finger' was prepared by solid-phase peptide synthesis. Fluorescence measurements showed that the tryptophan quantum yield was Zn(2+)-dependent, allowing a 1:1 a stoichiometry for the complex to be determined. The structure of the peptide was characterized using circular dichroic spectroscopy, which indicates that the peptide exhibits a random coiled conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc.     

Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of omega- and (omega-1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes

A peptide that inhibits the human cholesteryl ester transfer protein (CETP) was isolated from hog plasma by ultracentrifugation, two sequential column chromatographies and electroelution from gels. Molecular weight of the peptide was determined to be approximately 3 kDa on the SDS-PAGE. The peptide contained 28 amino acids with an identical sequence to the amino terminus of hog apolipoprotein-CIII except two amino acid residues: -Pro-Glu- at the fifth and sixth amino acids from the amino terminus in the isolated peptide, in contrast to -Leu-Leu- in hog apo-CIII. A peptide synthesized chemically according to the amino acid sequence of the peptide (designated P28) showed approximately the same degree of CETP inhibitory activity as the isolated peptide. Synthetic peptides with different number of amino acids were also tested for CETP inhibition. Among the peptides, the one with 20 amino acid residues (P20) from the amino terminus showed the highest inhibitory activity against the CETP. The peptide appeared to be associated with the hog high-density lipoproteins (HDL), as determined by immunoblot analysis using antibody against P28. The CETP-inhibitory activity of the peptide was examined in vivo using diet-induced hypercholesterolemic rabbits. When the peptide was injected into the rabbits (7-9 mg/kg body weight), approximately 75% CETP activity disappeared from the plasma in 1 h after the injection and the effect lasted up to 30 h. The inhibition of CETP in vivo led to a concomitant decrease in total plasma cholesterol level up to 30% and an increase in the level of HDL-cholesterol up to 32%. The cholesterol concentrations in the rabbit plasma gradually recovered to the initial level after 48 h.     

Genotype analysis of prepro-vasopressin signal peptide in vasopressin-producing and -non-producing lung tumors

A polymorphism in the nucleic acid sequence encoding the signal peptide of the human prepro-vasopressin (AVP) has been reported in an AVP producing small cell lung carcinoma (SCLC) cell line. The difference predicts expression in tumor cells of a variant signal peptide with Pro for Leu 11. To clarify whether this difference is required for AVP secretion from SCLC cells and/or reflects increased mutagenesis in malignant tumors, the exon encoding the signal peptide of prepro-AVP in two AVP producing SCLC and 9 non-producing lung tumors was amplified using polymerase chain reaction. The variant sequence was neither found by direct sequencing nor by restriction enzyme analysis. These results suggest that similar to the hypothalamus the normal signal peptide is functional in tumor cells and that the variant signal peptide is not a prerequisite for AVP secretion from SCLC cells.     

Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor

The islets of Langerhans contain four distinct endocrine cell types producing the hormones glucagon, insulin, somatostatin and pancreatic polypeptide. These cell lineages are thought to arise from a common, multipotential progenitor cell whose identity has not been well established. The pancreatic and intestinal hormone, peptide YY, has been previously identified in glucagon-producing cells in islets; however, transgenic mice expressing Simian Virus 40 large T antigen under the control of the peptide YY gene expressed the oncoprotein in beta, delta and pancreatic polypeptide cells, and occasionally developed insulinomas, suggesting relationships between peptide YY-producing cells and several islet cell lineages. The four established pancreatic islet cell types were examined for coexpression of peptide YY in islets of normal and transgenic mice throughout development. Peptide YY immunoreactivity was identified in the earliest endocrine cells in the fetal pancreas and was coexpressed in each islet cell type during development. Peptide YY showed a high degree of co-localization with glucagon- and insulin-producing cells in early pancreatic development, but by adulthood, peptide YY was expressed in less than half of the alpha cells and was no longer expressed in beta cells. Peptide YY was also coexpressed with somatostatin and pancreatic polypeptide when these cell types first appeared, but most delta and pancreatic polypeptide cells continued to express peptide YY throughout development. The use of conditions that distinguish peptide YY from the related peptides, pancreatic polypeptide and neuropeptide Y, as well as the ability of the peptide YY gene to direct expression of a reporter gene in islets of transgenic mice, establishes expression of peptide YY in the earliest pancreatic endocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of endogenous peptides eluted from the class I MHC molecule HLA-B7 determined by mass spectrometry and computer modeling

Microcapillary HPLC electrospray ionization tandem mass spectrometry was used to sequence 15 peptides eluted from HLA-B7. Sequence alignment implicated four peptide positions in specific interactions with the class I molecule, and their importance was confirmed using synthetic peptides. Because no crystal structure for HLA-B7 was available, computer-assisted modeling was used to understand novel aspects of peptide binding specificity and to accurately predict the effect of defined changes in peptide structure. The results demonstrate that mass-spectrometric sequencing coupled with computer-assisted modeling can be used in the absence of a crystal structure to make accurate predictions concerning requirements for peptide binding to class I molecules. These techniques may be valuable to predict or engineer T cell epitopes.     

Human interleukin-2-activated adherent natural killer cells recognize a conserved antigen found on tumor cells and protozoan parasites

Plastic adherent interleukin-2-activated human natural killer (NK) cells (ALAK) lyse many different histological types of tumor target cells. In order to effect their function as cytotoxic mediators of innate immunity, ALAKs may 'recognize' antigen(s) of protozoan parasites, select virus-infected cells and they may release certain cytokines in response to bacterial antigens. In the present study, we demonstrate that CD3-/CD56dim/CD16dim/monoclonal antibody 5C6bright human ALAKs bind to an antigenic determinant on tumor cells independent of target cell H-2 allotype expression. The conserved antigen was originally obtained from the protozoan Tetrahymena pyriformis, however it is also located on the membranes of many ALAK-sensitive tumor cells. The sequence of this protein, i.e. NK target antigen/NKTag, was previously deduced from cDNA. One ALAK cognate determinant of NKTag was identified by inhibition of cytotoxicity using NKTag-derived synthetic peptides. Biotinylated synthetic peptide [amino acids (aa) 58-74] bound to ALAKs, and synthetic peptides corresponding to this sequence inhibited ALAK lysis of U937 target cells. Inhibition effects of peptide binding were nonreversible. To determine the requirements for recognition by ALAKs of this antigenic determinant, the cognate peptide aa 55-74 was truncated to 17-, 14-, 10-, 7- and 6-mer lengths and tested for inhibition of cytotoxicity. All inhibited except the 6-mer. A possible mechanism of peptide inhibition of cytotoxicity following ALAK binding to an antigenic determinant was a requirement for recognition of one anchor peptide (arginine) and receptor occupancy by a minimum of five to six additional amino acids. In antibody-dependent cell-mediated cytotoxicity experiments, synthetic peptide (aa 68-74) inhibited ALAK killing of anti-H-2d-sensitized P815 targets. This same peptide also inhibited conventional lysis of nonsensitized P815 and IM-9 targets. However, the cognate synthetic peptide (aa 58-74) did not inhibit conjugate formation between ALAKs and U937 target cells. These data demonstrate that ALAK binding to a soluble monomeric peptide inhibited cytotoxicity. Peptide binding appeared to negatively regulate cytotoxicity, and the inhibitory effects following peptide binding were nonreversible. Effector:target cell conjugate formation was not affected by peptide binding, however, recognition was required because inhibition was specific for the amino acid sequence of the synthetic peptide.     

Gbeta subunit interacts with a peptide encoding region 956-982 of adenylyl cyclase 2. Cross-linking of the peptide to free Gbetagamma but not the heterotrimer

The region encoded by amino acids 956-982 of adenylyl cyclase 2 is important for Gbetagamma stimulation. Interactions of a peptide encoding the 956-982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with Gbetagamma subunits were studied. QEHA peptide was covalently attached to beta subunit of free Gbetagamma by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides cross-linked minimally. When Go was used, very little cross-linking was observed with GDP and EDTA, but upon activation by guanosine 5'-3-O-(thio)triphosphate and Mg2+, specific cross-linking of the QEHA peptide to Gbeta was observed. We conclude that beta subunits of G proteins contain effector interaction domains that are occluded by Galpha subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to Gbeta indicate that amino acids 75-165 of Gbeta may be involved in effector interactions.