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Phosphatidylcholine-polymer-coated plastic slides were utilized for the fabrication of peptide microarrays for cellular kinome analysis. According to the non-fouling features of the surface, the signal-to-noise ratio of the detection of phosphorylated peptides improved by about 100-fold from that of a peptide microarray fabricated on a glass slide blocked by a commercial BSA-based reagent. When the phosphatidylcholine-polymer-coated peptide microarray was applied to the analysis of the kinome of HCC827 cells, hyperactivation of c-Src and EGFR were successfully detected.

We previously reported that poly(N-isopropylacrylamide) grafted with Peptide 1 (-GLRRASLG) and poly(ethylene glycol) changed its physical properties in response to an intracellular protein phosphorylation signal, protein kinase A (PKA) (Katayama, Y. et al. (2001) Macromolecules 34, 905). In this study, we investigated the effect of changing peptide structure on the lower critical solution temperature (LCST) of peptide−polymer conjugates, before and after phosphorylation with PKA. For Peptide 2 (Ac-LRRASL-), which has a formal net charge of +2 at physiological pH, the LCST of the conjugate decreased on phosphorylation. In contrast, the LCSTs of the conjugates with Peptide 3 (-ALRRASLE) and Peptide 4 (Ac-DWDALRRASL-), which have neutral net charges, were greatly increased. This suggests that the LCST of the polymer was mainly governed by two factors:  the change in hydration around the polymer chain and the interpeptide electrostatic repulsion, resulting from phosphorylation. These polymers have potential for use as drug capsules that respond to cellular conditions.

We designed a peptide−polymer conjugate (CPCCtat) as a novel gene carrier that could control gene expression responding to the intracellular caspase-3 signal. This carrier consists of an uncharged main polymer chain and a cationic peptide side chain, which includes the substrate sequence of caspase-3 and the protein transduction domain sequence of HIV-1 Tat. In the present study, CPCCtat formed a tight complex with DNA through an electrostatic interaction, and in this state the gene expression was totally suppressed. In contrast, the complex disintegrated in the presence of caspase-3 due to cleavage of the cationic portion from CPCCtat. This event led to an activation of gene expression. Our results also indicate that the complex can be delivered into living cells due to the cell-permeable peptide side chain of CPCCtat. This intracellular signal-responsive system with CPCCtat will be useful for the cell-specific gene expression system.

Monodispersed dendritic poly(l-lysine)s (DPKs) of several generations were synthesized, and their characteristics as a gene transfection reagent were then investigated. The agarose gel shift and ethidium bromide titration assay proved that the DPKs of the third generation and higher could form a complex with a plasmid DNA, and the degree of compaction of the DNA was increased by the increasing number of the generation. The DPKs of the fifth and sixth generation, which have 64 and 128 amine groups on the surface of the molecule, respectively, showed efficient gene transfection ability into several cultivated cell lines without significant cytotoxity. In addition, the transfection efficiency of the DPK of the sixth generation was not seriously reduced even if serum was added at 50% of the final concentration into the transfection medium. Because we can strictly synthesize various DPK derivatives, which have several types of branch units, terminal cationic groups, and so on, they are expected to be a good object of study regarding the basic information on the detailed mechanism of gene transfection into cells. We also expect to be able to easily construct DPK-based functional gene carriers, e.g., DPKs modified by ligands such as a sugar chain, which can enable advanced gene delivery in vivo.

A 25-mer cationic peptide pleurocidin, isolated from the winter flounder, has broad antibacterial activity. To clarify the structure-activity relationship, its properties and biological activity were examined. CD measurements showed that pleurocidin took an α-helical structure in the presence of DOPC/DOPG (3 : 1, anionic) vesicles. Very weak hemolytic activity of pleurocidin was observed and its antibacterial activity was moderate. Tryptophan fluorescence shift measurements showed that pleurocidin interacted weakly with a neutral phospholipid, but strongly with an acidic phospholipid. The peptide exhibited weak dye-leakage activity for DOPC (neutral) vesicles and moderate activity for acidic vesicles. From experiments on dye-leakage activity and membrane translocation of the peptide, it seemed likely that pleurocidin, like magainin 2, forms pores in the lipid membrane. A study of amino acid substitution in pleurocidin revealed that α-helicity, rather than hydrophobicity, affects the properties and activity of the peptide.

Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218-222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPbeta, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). PMA induced expression of RPTPbeta mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D(3) and interferon-gamma, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPbeta mRNA, whereas 1. 2% Me(2)SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPbeta antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPbeta. Double immunostaining studies also indicated that newly expressed RPTPbeta colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPbeta cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPbeta, a protein that functions as the VacA receptor.

For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic α-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH₂) obtained from wasp venom and an α-helix model peptide (LARL-LARL-LARL-NH₂). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide.

Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA. Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry. Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-beta(1-3)-N-acetylgalactosamine and galactose-beta(1-4)-N-acetylglucosamine. p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively. These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPbeta/PTPzeta); in agreement, p250 reacted with anti-human RPTPbeta monoclonal antibody. Immunoprecipitation with anti-human RPTPbeta antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPbeta bound native, but not denatured, VacA. Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPbeta.

Recent genetic and molecular biological analyses have revealed many forms of inherited channelopathies. Homozygous ataxic mice, tottering (tg) and leaner (tgla) mice, have mutations in the P/Q-type Ca2+ channel alpha1A subunit gene. Although their clinical phenotypes, histological changes, and locations of gene mutations are known, it remains unclear what phenotypes the mutant Ca2+ channels manifest, or whether the altered channel properties are the primary consequence of the mutations. To address these questions, we have characterized the electrophysiological properties of Ca2+ channels in cerebellar Purkinje cells, where the P-type is the dominant Ca2+ channel, dissociated from the normal, tg, and tgla mice, and compared them with the properties of the wild-type and mutant alpha1A channels recombinantly expressed with the alpha2 and beta subunits in baby hamster kidney cells. The most striking feature of Ca2+ channel currents of mutant Purkinje cells was a marked reduction in current density, being reduced to approximately 60 and approximately 40% of control in tg and tgla mice, respectively, without changes of cell size. The Ca2+ channel currents in the tg Purkinje cells showed a relative increase in non-inactivating component in voltage-dependent inactivation. Besides the same change, those of the tgla mice showed a more distinct change in voltage dependence of activation and inactivation, being shifted in the depolarizing direction by approximately 10 mV, with a broader voltage dependence of inactivation. In the recombinant expression system, the tg channel with a missense mutation (P601L) and one form of the two possible tgla aberrant splicing products, tgla (short) channel, showed a significant reduction in current density, while the other form of the tgla channels, tgla (long), had a current density comparable to the normal control. On the other hand, the shift in voltage dependence of activation and inactivation was observed only for the tgla (long) channel. Comparison of properties of the native and recombinant mutant channels suggests that single tottering mutations are directly responsible for the neuropathic phenotypes of reduction in current density and deviations in gating behavior, which lead to neuronal death and cerebellar atrophy.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (46, Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH2, and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 46 had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 46 and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide−DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.

Polycationic reagents such as cationic lipids and poly-L-lysine are widely used for gene transfer into cells in vitro and show promise as vectors for in vivo gene therapy applications as nonviral gene transfer techniques. We have developed a novel transfection method using cationic amphiphilic alpha-helical oligopeptides with repeated sequences. Oligopeptide has the advantages of being easily designed and modified because of its simple structure. In this study, we synthesized five kinds of peptides of which the total chain length and the width of the hydrophobic region were changed. The binding of the peptides to plasmid DNA was evaluated by agarose gel electrophoresis. It was found that the long and/or hydrophobic peptides can strongly bind to the DNA. The formation of large aggregates with a 0.5-5-microm diameter, which consisted of the long peptides and the DNA, was observed by electron microscopy. The transfection abilities of the peptides were determined by the expression of luciferase from its cDNA in COS-7 cells. The long peptides showed high transfection abilities. As a result, it could be said that the transfection ability of these peptides was parallel to their ability to form aggregates with DNA. Furthermore, the transfection ability was increased by the addition of chloroquine in the transfection procedure. This result indicated that the internalization of the peptide-DNA aggregates would be mediated by the endocytosis pathway.

We characterized the electrophysiological properties of calcium channels in cultured rat hippocampal neurones using ω-agatoxin-TK (ω-Aga-TK) and compared them with those of the P-type channel and the BI (α1A) channel which resembles the Q-type channel. Two types of ω-Aga-TK-sensitive calcium channels were detected in hippocampal neurones. The first type showed slow inactivation, high sensitivity to ω-Aga-TK and low reversibility from ω-Aga-TK-induced block, resembling the P-type channel. The second type showed fast inactivation, low sensitivity to ω-Aga-TK and high reversibility from ω-Aga-TK-induced block, resembling the Q-type channel. The glutamatergic synaptic transmission showed low sensitivity to ω-Aga-TK and high reversibility from ω-Aga-TK-induced block. These results suggest that the second type of calcium channel (Q-type-like) plays a prominent role in the hippocampal synaptic transmission.

A cDNA encoding the smaller subunit (P52) of mitochondrial processing protease was isolated from a rat liver cDNA library, using cDNA fragment for yeast MAS1 as the probe. The deduced amino acid sequence is highly homologous to those of PEP from Neurospora crassaand MAS1 from Saccharomyces cerevisiae. After in vitrotranscription and translation, the precursor peptide was imported into isolated rat liver mitochondria and processed to its mature form.

In the course of purification of ω-Aga-IVA, a specific P-type calcium channel blocker, from the venom of Agelenopsis aperta we discovered a novel peptide. This peptide, named ω-agatoxin Tsukuba (ω-Aga-TK), also blocked P-type channels and was twelve times more abundant in the venom than ω-Aga-IVA. ω-Aga-TK was purified to homogeneity by a two-step reverse-phase HPLC procedure. Its amino acid sequence is 71% identical to that of ω-Aga-IVA. ω-Aga-TK has a negatively charged N-terminus, whereas ω-Aga-IVA has a positively charged one. Electrophysiological data indicate that ω-Aga-TK is a potent and selective inhibitor of P-type channels.Copyright 1993, 1999 Academic Press

Plasmids containing the BI (α1) cDNA with the dihydrofolate reductase (DHFR) gene, skeletal muscle α2-subunit cDNA with the neomarker gene, and β-subunit cDNA were co-transfected into baby hamster kidney (BHK) cells. BHK cells lack endogenous calcium channel activity. Twenty percent of the methotrexate (MTX) and G418 resistant clones were found to express calcium channel activity using the patch-clamp technique. A single clone, BHKBI147, demonstrated stable electrophysiological characteristics over 20 passages. Ca2+currents of the BI channel in BHKBL147 cells were largely blocked by a specific P-type blocker, ω-AgaIVA, with an IC50of 150 nM. Unlike the BI channel, Ca2+currents of cardiac L-type channels expressed in BHK cells were completely blocked by the L-type antagonist, nifedipine, with an IC50of 56 nM.

omega-Agatoxin-TK (omega-Aga-TK), a 48-amino-acid peptide isolated from the venom of the funnel web spider (Agelenopsis aperta), is a selective and potent inhibitor of P-type calcium channels in the nervous system. We have synthesized a peptide that has the amino acid sequence identified for native omega-Aga-TK. The synthetic omega-Aga-TK, however, showed 80-90-fold less potent inhibition of P-type calcium channels, compared with native omega-Aga-TK. Enantiomer analysis of native omega-Aga-TK revealed D-serine at position 46, and synthetic omega-[D-Ser46]Aga-TK had the same potency as native omega-Aga-TK for blocking P-type calcium channels in cultured cerebellar Purkinje neurons. Two peptide fragments of omega-Aga-TK, namely omega-Aga-TK(1-43) and the carboxyl-terminal peptide fragment omega-Aga-TK(44-48), did not produce any significant inhibition of P-type calcium channels or interfere with the blockade of the channels elicited by native omega-Aga-TK. Molecular dynamics calculations showed that the carboxyl-terminal, six-amino-acid peptide of omega-Aga-TK containing D-Ser46 assumes a different conformation than does the peptide containing L-Ser46. These results suggest that the specific conformation of the carboxyl-terminal region of omega-Aga-TK, particularly the configuration of Ser46, together with a beta-sheet structure formed by four disulfide bonds, might be essential for blockade of P-type calcium channels.