Naturally occurring CD4+ CD25+ cells in modulating immune response to administered coagulation factor VIII in factor VIII-deficient mice.

CD4+ CD25+ T regulatory (Treg) cells are critical mediators of peripheral self-tolerance and immune homeostasis. In this study, we characterized the ability of naturally occurring CD4+ CD25+ cells from the wild-type mice to modulate the immune response to administered coagulation factor VIII (FVIII) in FVIII-deficient mice. For the cell therapy, CD4+ CD25+ cells and CD4+ CD25- cells were purified from the spleens of wild-type normal mice and administered to FVIII-deficient mice prior to four injections of recombinant FVIII (rFVIII). The titre of FVIII antibodies and antibodies with inhibitory activity against FVIII was lower in the mice treated with natural CD4+ CD25+ cells or CD4+ CD25- cells compared with the mice treated only with rFVIII. We also demonstrated that CD4+ CD25- cells could differentiate to acquire the Treg phenotype expressing CD25 and FoxP3 if stimulated in vitro. These observations provide evidence that Treg cells can be used for designing cell therapy for controlling the immune response to FVIII.

Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Cargo delivery kinetics of cell-penetrating peptides.

A diversity of cell-penetrating peptides (CPPs), is known, but so far the only common denominator for these peptides is the ability to gain cell entry in an energy-independent manner. The mechanism used by CPPs for cell entry is largely unknown, and data comparing the different peptides are lacking. In order to gain more information about the cell-penetrating process, as well as to quantitatively compare the uptake efficiency of different CPPs, we have studied the cellular uptake and cargo delivery kinetics of penetratin, transportan, Tat (48-60) and MAP (KLAL). The respective CPPs (labelled with the fluorescence quencher, 3-nitrotyrosine) are coupled to small a pentapeptide cargo (labelled with the 2-amino benzoic acid fluorophore) via a disulfide bond. The cellular uptake of the cargo is registered as an increase in fluorescence intensity when the disulfide bond of the CPP-S-S-cargo construct is reduced in the intracellular milieu. Our data show that MAP has the fastest uptake, followed by transportan, Tat(48-60) and, last, penetratin. Similarly, MAP has the highest cargo delivery efficiency, followed by transportan, Tat (48-60) and, last, penetratin. Since some CPPs have been found to be toxic at high concentration, we characterized the influence of CPPs on cellular 2-[(3)H]deoxyglucose-6-phosphate leakage. Measurements on this system show that the membrane-disturbing potential appears to be correlated with the hydrophobic moment of the peptides. In summary, the yield and kinetics of cellular cargo delivery for four different CPPs has been quantitatively characterized.

Deletion analogues of transportan.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

Targeting of cancer-related proteins with PNA oligomers.

Aberrant gene expression is characteristic to all cancer cells and pathophysiology in general. Selective inhibition of constitutively elevated expression of oncogenes provides an opportunity to hinder the proliferation of malignant cells. Small synthetic molecules that specifically interfere with transcription and/or translation have great potential as anticancer drugs. Currently first-generation antisense oligonucleotides are widely used to inhibit the oncogene expression. The second generation of antisense agents have been studied mainly in vitro. One of these agents, peptide nucleic acid (PNA) is an oligonucleotide mimic with a non-charged achiral polyamide backbone to which the nucleobases are linked. PNA oligomers bind tightly to complementary DNA or RNA and are very stable in biological fluids. PNA can inhibit transcription and translation of target genes by specifically hybridizing to DNA or mRNA. The in vitro experiments showing inhibition of target protein expression by PNA have been followed by the first successful applications of PNA as an antisense agent in cultured cells and also in vivo. Hopefully this will lead to a wider use of PNA in the studies of cancer biology and therapy.

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes.

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.

Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.

PNA oligomers as tools for specific modulation of gene expression.

Small synthetic molecules that can specifically inhibit translation and/or transcription have shown great promise as potential antisense/antigene drugs. Peptide nucleic acid (PNA), an oligonucleotide mimic, has a non-charged achiral polyamide backbone to which the nucleobases are attached. PNA oligomers are extremely stable in biological fluids and they specifically hybridise to DNA or RNA in a complementary manner, forming very strong heteroduplexes. Some of the mRNAs have yet undetermined and possibly long half-lives, successful down regulation of gene expression by antisense oligonucleotides (ON) requires that the antisense agent is long lived. PNA fulfils this requirement better than phosphodiester or phosphorothioate ONs. PNA can inhibit transcription and translation of respective genes by tight binding to DNA or mRNA. First in vitro experiments to specifically down regulate protein expression by PNA have been followed by successful antisense and antigene application of PNA oligomers in vivo. This review discusses the principles of the in vitro and in vivo use of PNA oligonucleotides.

A galanin-mastoparan chimeric peptide activates the Na+,K(+)-ATPase and reverses its inhibition by ouabain.

The effect of the neuropeptide galanin, the wasp venom toxin amphiphilic peptide toxin mastoparan and the chimeric peptide, galparan, consisting of N-terminal 13 amino acids of neuropeptide galanin linked at C-terminus to mastoparan amide (and its inactive analog Mas17) on the activity of Na+,K(+)-ATPase has been studied. Mastoparan inhibits the activity of the Na+,K(+)-ATPase with IC50 = 7.5 microM and also reduces the cooperativity for Na+ and K+, respectively, while galanin has no effect on the Na+,K(+)-ATPase activity. The chimeric peptide, galanin(1-13)-mastoparan amide (galparan), exhibits biphasic interaction with Na+,K(+)-ATPase, it activates the enzyme at maximal stimulating concentration of 4 microM followed by inhibition of the enzyme with IC50 of 100 microM. At maximum stimulating concentration (4 microM), galparan partly reduces the cooperativity only for Na+ and it also counteracts the inhibitory effect of oubain on Na+,K(+)-ATPase. Galparan's stimulatory effect was influenced by ATP. The chimeric peptide [19Lys,26Leu]-galparan, containing the inactive analog of mastoparan (Mas17), has no effects on rat brain Na+,K(+)-ATPase activity. Both chimeric peptides galparan and [19Lys,26Leu]-galparan are high-affinity galanin receptor ligands with IC50 of 6.4 nM and 0.71 nM, respectively, while galanin (1-13) and mastoparan alone have significantly lower affinity for the galanin receptor, IC50 of 125 nM and 1 microM, respectively. The ability of chimeric peptides to bind to galanin receptors does not correlate with their effects on the Na+,K(+)-ATPase.

Galparan induces in vivo acetylcholine release in the frontal cortex.

The chimeric peptide galparan (galanin(1-13)-mastoparan) induced the in vivo release of acetylcholine in the frontal cortex of rats when injected intracerebroventricularly, i.c.v. The ACh-releasing effects of galparan are reversible, dose-dependent, and not exerted at galanin receptors or at sites where mastoparan acts. Pertussis toxin pretreatment (i.c.v.) of the rats for 96 h prior to injection of galparan or of mastoparan completely prevented the ACh-releasing effects of both galparan and mastoparan. It appears that galparan acts at a novel site in the release of ACh in the cerebral cortex in vivo.

Production and characterization of monoclonal antibodies against class theta glutathione S-transferase T1-1.

The recently discovered human class theta glutathione S-transferase T1-1 (GSTT1-1) is responsible for the GSH-dependent detoxification of naturally occurring monohalomethanes. The detoxifying role of GSTT1-1 has not been investigated in cancer susceptibility and the polymorphism of the protein is unknown in different populations. The purpose of our work was to produce a panel of mouse monoclonal antibodies (MAbs) that could bind to different regions of the GSTT1-1 protein and would help us select suitable MAbs for Western blot analyses and immunohistochemistry, and develop an ELISA assay for detection of GSTT1-1 in whole blood. Six highly specific MAbs were generated against GSTT1-1. Out of six MAbs, one was able to recognize only the native form of the enzyme and possesses two binding sites on the dimeric GSTT1-1 molecule. The other five MAbs bind to both native and denatured GSTT1-1 enzyme in direct and antigen capture ELISA or Western blot. The antibodies recognize at least four different epitopes on the GSTT1-1 molecule. Using MAbs 4G1 and 2D8, a sensitive ELISA assay for determination of GSTT1-1 in whole blood was developed.

Cell penetration by transportan.

Transportan is a 27 amino acid-long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin and mastoparan in the carboxyl terminus, connected via a lysine. Transportan is a cell-penetrating peptide as judged by indirect immunofluorescence using N epsilon13-biotinyl-transportan. The internalization of biotinyl-transportan is energy independent and takes place efficiently at 37 degrees, 4 degrees, and 0 degrees C. Cellular uptake of transportan is probably not mediated by endocytosis, since it cannot be blocked by treating the cells with phenylarsine oxide or hyperosmolar sucrose solution and is nonsaturable. The kinetics of internalization was studied with the aid of the 125I-labeled peptide. At 37 degrees C, the maximal intracellular concentration is reached in about 20 min. The internalized transportan is protected from trypsin. The cell-penetrating ability of transportan is not restricted by cell type, but seems to be a general feature of this peptide. In Bowes' melanoma cells, transportan first localizes in the outer membrane and cytoplasmatic membrane structures. This is followed by a redistribution into the nuclear membrane and uptake into the nuclei where transportan concentrates in distinct substructures, probably the nucleoli.

13C and 15N NMR and time-resolved fluorescence depolarization study of bovine carbonic anhydrase--4-methylbenzenesulfonamide complex.

The influence of the binding of the high-affinity inhibitor, 4-methylbenzenesulfonamide, to the active site of bovine carbonic anhydrase B was studied by 15N- and 13C-NMR spectroscopy. The rotational correlation time dependence on temperature and concentration of the complex was determined by time-resolved fluorescence depolarization measurements. Our experiment provides evidence that the stoichiometry of the interaction of 4-methylbenzenesulfonamide with carbonic anhydrase B is 1:1 and the inhibitor is bound in anionic form. The 15N-NMR relaxation parameters confirm our previous conclusions about the presence of librational motions in the active site of carbonic anhydrase and indicate that the internal motion in the enzyme-inhibitor complex is more restricted than the backbone motion in the uncomplexed native enzyme.

Separation of the rotational contribution in fluorescence correlation experiments.

The theory of fluorescence correlation spectroscopy is reexamined with the aim of separating the contribution of rotational diffusion. Under constant excitation, fluorescence correlation experiments are characterized by three polarizations: one of the incident beam and two of the two photon detectors. A set of experiments of different polarizations is proposed for study. From the results of the experiments the isotropic factor of the fluorescence intensity correlation functions can be determined, which is independent of the rotational motion of the sample molecule. This function can be used to represent each fluorescence intensity correlation function as the product of the isotropic and the rotational factors. The theory is illustrated by an experiment in which rotational diffusion of porcine pancreatic lipase labeled with Texas Red was observed Texas Red is a label that allows precise fluorescence correlation experiments even in the nanosecond time range.

Fluorescence correlation spectroscopy in the nanosecond time range: rotational diffusion of bovine carbonic anhydrase B.

A fluorescence correlation experiment for measurement of rotational diffusion in the nanosecond time scale is described. Using this method, the rotational diffusion coefficient of bovine carbonic anhydrase B labelled with tetramethylrhodamine isothiocyanate was estimated to be Dr = (1.14 +/- 0.15) X 10(7) s-1 at 22 degrees C. The experiment is based on a cw argon ion laser, a microfluorometer with local solution flow inside the sample cell, and two photon detectors. The fluorescence intensity autocorrelation function in the nanosecond time range is computed with the help of a time-to-amplitude converter and a multichannel pulse-amplitude analyser.

Purification, characterization and tissue distribution of human class theta glutathione S-transferase T1-1.

A high activity glutathione S-transferase T1-1 (GSTT1-1) towards dichloromethane was isolated from human liver cytosol and purified to homogenity in 18.5% yield with a purification factor of 4400-fold. The GSTT1-1 was also isolated from erythrocytes, but the enzyme activity decreased rapidly in the final stages of purification. The purified GSTT1-1-s were homo-dimeric enzymes with a subunit M1 value 25,300 and pI 6 64, as confirmed by SDS-PAGE, IEF and Western blot analysis. The N-terminal amino acid sequences of GSTT1-1 from liver and red blood cells, analyzed up to the 12th amino acid, were identical. Immunoblot analysis revealed that GSTT1-1 was also present in lung, kidney, brain, skeletal muscle, heart, small intestine and spleen, but not in lymphocytes.

Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain. Comparison with the penetratin peptide.

Cellular translocation into a human Bowes melanoma cell line was investigated and compared for penetratin and pIsl, two peptides that correspond to the third helices of the related homeodomains, from the Antennapedia transcription factor of Drosophila and the rat insulin-1 gene enhancer protein, respectively. Both biotinylated peptides internalized into the cells with similar efficacy, yielding an analogous intracellular distribution. When a large cargo protein, 63 kDa avidin, was coupled to either peptide, efficient cellular uptake for both the peptide-protein complexes was observed. The interactions between each peptide and SDS micelles were studied by fluorescence spectroscopy and acrylamide quenching of the intrinsic tryptophan (Trp) fluorescence. Both peptides interacted strongly and almost identically with the membrane mimicking environment. Compared to penetratin, the new transport peptide pIsl has only one Trp residue, which simplifies the interpretation of the fluorescence spectra and in addition has a native Cys residue, which may be used for alternative coupling reactions of cargoes of different character.

Differential regulation of GTPase activity by mastoparan and galparan.

The chimeric peptide galparan, composed of galanin (1-13) in the N-terminus and mastoparan in the C-terminus, was recently designed and synthesized. The effect of galparan on GTPase activity of rat brain cortical membranes was studied in comparison with the effect of mastoparan and galanin. GTPase was activated by mastoparan but it was noncompetitively inhibited by galparan, while no effect of galanin and galanin (1-13) was found in this tissue. EC50 of 12.1 +/- 2.1 microM and Hill coefficient of 2.1 +/- 0.6 was calculated for galparan from a dose-response curve and Ki of 19.1 +/- 0.3 microM was obtained by fitting the experimental data to the Michaelis-Menten equation valid in the presence of noncompetitive inhibitor. Mastoparan reversed the effect of galparan in a fully competitive manner while benzalkonium chloride did not prevent the inhibition of GTPase activity by galparan. Pertussis-toxin-catalyzed ribosylation of G proteins from rat brain cortical membranes resulted in 15% lower basal GTPase activity of our preparation but did not alter the parameters of the dose-response curve for galparan inhibition. The rate of GTP gamma S binding to G proteins from rat brain cortical membranes was not influenced by galparan. CD spectra revealed predominantly antiparallel beta-structure and unordered secondary structure of galparan in the buffer solution, while in the presence of lipid vesicles it adopted a higher amount of alpha-helix. Critical micelle concentration of galparan in buffer solution of 22 microM was determined. It is suggested that the reversal of GTPase activation by mastoparan to inhibition by galparan is due to different loci of action of these two peptides on G proteins.

Conformational analysis of galanin using end to end distance distribution observed by Förster resonance energy transfer.

The structural dynamics of the flexible neuropeptide galanin in solution were studied by Förster resonance energy transfer measurements at different temperatures by time-resolved fluorescence spectroscopy to determine its conformational heterogeneity. Endogenous tryptophan at position 2 acted as the fluorescent donor and the non fluorescent acceptor dinitrophenyl or the fluorescent acceptor dansyl were selectively attached to lysine 25 in porcine galanin. The coexistence of different structures of the neuropeptide galanin in trifluoroethanol solution was revealed by the model independent analysis of the distribution of relaxation times from the time-resolved resonance energy transfer data. Multiple conformational states are reflected by distinct end-to-end distance populations. The conformations differ in mean donor-acceptor distance by about 15, and are consistent with the extended and folded backbone conformations of two alpha-helical regions separated by a flexible hinge. The effect that the labelling of galanin has on binding to the receptor was also evaluated. DNP-galanin showed the same high affinity to galanin receptors as unlabelled galanin, whereas DNS-galanin had significantly reduced affinity.