A simple, rapid, and sensitive system for the evaluation of anti-viral drugs in rats.

The lack of small animal models for the evaluation of anti-human immunodeficiency virus type 1 (HIV-1) agents hampers drug development. Here, we describe the establishment of a simple and rapid evaluation system in a rat model without animal infection facilities. After intraperitoneal administration of test drugs to rats, antiviral activity in the sera was examined by the MAGI assay. Recently developed inhibitors for HIV-1 entry, two CXCR4 antagonists, TF14016 and FC131, and four fusion inhibitors, T-20, T-20EK, SC29EK, and TRI-1144, were evaluated using HIV-1(IIIB) and HIV-1(BaL) as representative CXCR4- and CCR5-tropic HIV-1 strains, respectively. CXCR4 antagonists were shown to only possess anti-HIV-1(IIIB) activity, whereas fusion inhibitors showed both anti-HIV-1(IIIB) and anti-HIV-1(BaL) activities in rat sera. These results indicate that test drugs were successfully processed into the rat sera and could be detected by the MAGI assay. In this system, TRI-1144 showed the most potent and sustained antiviral activity. Sera from animals not administered drugs showed substantial anti-HIV-1 activity, indicating that relatively high dose or activity of the test drugs might be needed. In conclusion, the novel rat system established here, "phenotypic drug evaluation", may be applicable for the evaluation of various antiviral drugs in vivo.

Bioorganic synthesis of a recombinant HIV-1 fusion inhibitor, SC35EK, with an N-terminal pyroglutamate capping group.

The bioorganic synthesis of an end-capped anti-HIV peptide from a recombinant protein was investigated. Cyanogen bromide-mediated cleavage of two Met-Gln sites across the target anti-HIV sequence generated an HIV-1 fusion inhibitor (SC35EK) analog bearing an N-terminal pyroglutamate (pGlu) residue and a C-terminal homoserine lactone (Hsl) residue. The end-capped peptide, pGlu-SC35EK-Hsl, had similar bioactivity and biophysical properties to the parent peptide, and an improved resistance to peptidase-mediated degradation was observed compared with the non-end-capped peptide obtained using standard recombinant technology.

Bioorganic synthesis of end-capped anti-HIV peptides by simultaneous cyanocysteine-mediated cleavages of recombinant proteins.

Bioorganic synthesis of N- and C-terminal end-capped peptides by two simultaneous S-cyanocysteine-mediated cleavages of recombinant proteins is described. This approach is demonstrated in the preparation of anti-HIV fusion inhibitory peptides.

Sugar-dependent photodynamic effect of glycoconjugated porphyrins: a study on photocytotoxicity, photophysical properties and binding behavior to bovine serum albumin (BSA).

The photocytotoxicity of four glycoconjugated porphyrins, namely 5,10,15,20-tetrakis[4-(beta-D-glucopyranosyloxy)phenyl]porphyrin (p-1a), 5,10,15,20-tetrakis[4-(beta-D-galactopyranosyloxy)phenyl]porphyrin (p-1b), 5,10,15,20-tetrakis[4-(beta-D-xylopyranosyloxy)phenyl]porphyrin (p-1c) and 5,10,15,20-tetrakis[4-(beta-D-arabinopyranosyloxy)phenyl]porphyrin (p-1d), was evaluated in HeLa cells in the concentration range from 1 to 7 microM using a light dose of 16 J x cm(-2) with a wavelength greater than 500 nm. The photocytotoxicity depends on the sugar moieties, and increases in the order of p-1d

A synthetic peptide corresponding to residues 301-320 of human Wnt-1 promotes PC12 cell adhesion and hippocampal neural stem cell differentiation.

Wnt signaling cascades play a crucial role in the maintenance of stem cell niches in many tissues as well as in embryonic patterning and cell-fate determination. Wnt signaling pathways have been well studied; however, the precise binding mechanism of Wnt protein to its receptor has not yet been clarified. Here we show the design and synthesis of seven novel peptide candidates for a receptor-binding site of human Wnt-1 based on its hydrophilicity and beta-turn profiles. Among these Wnt-derived peptides, only WP7, which corresponds to residues 301-320 of human Wnt-1, bound to the soluble receptor for Wnt-1, mouse Frizzled-1/Fc chimera, promoted PC12 cell adherence, increased level of cytosolic beta-catenin in PC12 cells, and induced adhesion and neuronal differentiation of hippocampal neural precursor cells. These results suggest that residues 301-320 of human Wnt-1 is one of the receptor-binding sites and that WP7 may activate the canonical Wnt pathway. When combined with an appropriate matrix, the action of this Wnt-derived peptide, WP7, can be limited to within a location, and therefore could be useful in the regeneration of many tissues, without fear of tumor generation.

Sugar-dependent aggregation of glycoconjugated chlorins and its effect on photocytotoxicity in HeLa cells.

In order to explore the influence of the sugar moieties of glycoconjugated chlorins on the photocytotoxicity, we studied the photochemical properties of four glycoconjugated chlorins in aqueous media such as cytoplasm and the concentration dependence of photocytotoxicity in HeLa cells. In phosphate-buffered saline, the fluorescence intensities of 5,10,15,20-tetrakis[3-(beta-D-glucopyranosyloxy)phenyl]chlorin (m-1a) and 5,10,15,20-tetrakis[3-(beta-D-galactopyranosyloxy)phenyl]chlorin (m-1b), i.e., chlorins having hexose groups, were about 2-fold greater than those of 5,10,15,20-tetrakis[3-(beta-d-xylopyranosyloxy)phenyl]chlorin (m-1c) and 5,10,15,20-tetrakis[3-(beta-d-arabinopyranosyloxy)phenyl]chlorin (m-1d), i.e., chlorins having pentose groups, owing to a sugar-dependent difference of aggregation behavior. While no cytotoxicity was found in the dark, the highest photocytotoxicity was shown by m-1a (82% inhibition) in HeLa cells. This was higher than those of m-1b, m-1c, m-1d and tetraphenylporphyrin tetrasulfonic acid. The glycoconjugated chlorins except for m-1b appeared to be distributed diffusely throughout the cytoplasm. Among the four photosensitizers, m-1a showed the highest intensity in confocal fluorescence images, in agreement with the in vitro photocytotoxicity results. For m-1c, no photocytotoxicity was found at drug concentrations from 0.2 to 0.04 microM. Hence, sugar-dependent aggregation is not the major reason for the unexpected lack of efficacy of m-1c, which is uptaken efficiently by HeLa cells. For the glycoconjugated chlorins, these results suggest the biological aspects of sugar moiety play much crucial role rather than chemical aspects.

Synthetic peptides corresponding to ligand-binding region of death receptors, DR5, Fas, and TNFR, specifically inhibit cell death mediated by the death ligands, respectively.

Apoptosis as well as cell growth and cell differentiation play an important role in the maintenance of homeostasis in multicellular organisms. Disruption of apoptosis causes serious diseases, such as cancer, chronic inflammatory diseases, and autoimmune diseases; therefore, the control of apoptosis is one of the most promising therapeutic approaches to these apoptosis-disrupted diseases. Apoptosis is mediated by soluble factors, which belong to the TNF superfamily, such as TNF-alpha, FasL, and TRAIL. Here, we report that we deduced ligand-binding domains based on the structure of apoptosis ligand-receptor complex, and the synthetic peptide corresponding to residues 91-102 of DR5 indeed showed specific binding to TRAIL molecule and inhibited TRAIL-induced cell death both in L929 cells and in HeLa cells. The other death receptor-derived peptides, which are the corresponding regions of TNFR1 and Fas, also showed specific binding to TNF-alpha and FasL and inhibited the ligand-induced cell death, respectively. These results suggest that the position of the ligand-binding region is conserved among these death-receptor family members, whereas the primary amino acid sequence determines ligand specificity.

Promotion of neurite outgrowth from fetal hippocampal cells by TNF-alpha receptor 1-derived peptide.

Cytokines such as tumor necrosis factor-alpha (TNF-alpha), FasL, and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis or inflammation through binding to their specific receptors, TNFR1, Fas, and DR5, respectively. We have previously reported ligand-binding and cell death-inhibiting synthetic peptides, which were designed based on the crystal structure of a ligand-receptor complex and the homology of the amino acid sequence among the death receptor family members. Here we show that, among these death receptor-derived peptides, the TNFR1-derived peptide specifically arrested cell proliferation and promoted cell adhesion of fetal rat (E16) hippocampal cells, and promoted neurite outgrowth from hippocampus-derived neurospheres cultured with the addition of the peptide or cultured on a peptide-coated surface. Furthermore, among these death receptor-derived peptides, marked neurite outgrowth was observed only when the neurospheres were cultured on a TNFR1-derived peptide-conjugated covalently cross-linked alginate gel. The neurites from the neurospheres positively immunostained with an antibody against neurofilaments. These results suggest that the TNFR1-derived peptide promotes neuronal differentiation of the hippocampal neural stem cells and the TNFR1-derived peptide-conjugated covalently cross-linked alginate gel may be a useful material for assisting neural stem cell transplantation.

The aromatic amino acid residues of tumor necrosis factor receptor-1-derived peptide are important for promoting differentiation of neural stem cells.

Neural stem cells have the self-renewal capacity and the ability to differentiate into all types of nerve cells. We previously reported that the tumor necrosis factor receptor-1-derived peptide promotes neural differentiation of fetal rat hippocampal neural stem cells. The tumor necrosis factor receptor-1-derived peptide contains six aromatic amino acid residues among its 14 amino acid residues. To clarify the role of these aromatic amino acid residues in the action of tumor necrosis factor receptor-1-derived peptide on neural stem cells, we synthesized mutant peptides, in which aromatic residues were substituted with alanine, and we assessed their effects. Substitution of the tyrosine residue at position 103 (Y(103)) or 106 (Y(106)), the tryptophan residue at position 107 (W(107)), or the phenylalanine residue at position 112 (F(112)) or 115 (F(115)), decreased the ability of the peptide to promote neurite outgrowth of neural stem cells depending on their concentration. These data suggest that although all five aromatic amino acid residues mediate the action of the tumor necrosis factor receptor-1-derived peptide, their order of importance in this activity is F(115) > Y(103) > W(107) > Y(106) and F(112).

The biodegradability of poly(Pro-Hyp-Gly) synthetic polypeptide and the promotion of a dermal wound epithelialization using a poly(Pro-Hyp-Gly) sponge.

Collagens are widely used in medical applications, but animal-derived collagens have several drawbacks, such as low thermal stability, nonspecific cell attachment, and susceptibility to contamination by infectious pathogens, such as prions, which may transfect humans. We have previously reported the chemical synthesis of polypeptides consisting of a Pro-Hyp-Gly sequence and the high thermostability of their triple-helical structure. To clarify the biomaterial characteristics of the poly(Pro-Hyp-Gly) polypeptide, we assessed its biodegradability and its capability for skin regeneration. Eight weeks after implantation, a poly(Pro-Hyp-Gly) freeze-dried sponge embedded subcutaneously into a rat dorsal area degraded at the same rate as Terudermis, which is made from bovine type I atelocollagen and is used as an artificial dermis. Surprisingly, compared with Terudermis, the poly(Pro-Hyp-Gly) sponge significantly promoted epithelialization of a full-thickness wound on a rabbit's ear pad. This chemically synthesized polypeptide may be useful as a scaffold for tissue engineering and tissue regeneration.

Promotion of Neurite Outgrowth from Fetal Hippocampal Cells by TNF-α Receptor 1-Derived Peptide.

Cytokines such as tumor necrosis factor-α (TNF-α), FasL, and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis or inflammation through binding to their specific receptors, TNFR1, Fas, and DR5, respectively. We have previously reported ligand-binding and cell death-inhibiting synthetic peptides, which were designed based on the crystal structure of a ligand-receptor complex and the homology of the amino acid sequence among the death receptor family members. Here we show that, among these death receptor-derived peptides, the TNFR1-derived peptide specifically arrested cell proliferation and promoted cell adhesion of fetal rat (E16) hippocampal cells, and promoted neurite outgrowth from hippocampus-derived neurospheres cultured with the addition of the peptide or cultured on a peptide-coated surface. Furthermore, among these death receptor-derived peptides, marked neurite outgrowth was observed only when the neurospheres were cultured on a TNFR1-derived peptide-conjugated covalently cross-linked alginate gel. The neurites from the neurospheres positively immunostained with an antibody against neurofilaments. These results suggest that the TNFR1-derived peptide promotes neuronal differentiation of the hippocampal neural stem cells and the TNFR1-derived peptide-conjugated covalently cross-linked alginate gel may be a useful material for assisting neural stem cell transplantation.

Induction of apoptosis in HL-60 cells by photochemically generated hydroxyl radicals.

Reactive oxygen species (ROS), especially hydroxyl radicals are postulated to mediate apoptosis of the cell. Here we demonstrate that hydroxyl radicals generated selectively by photolysis of a photo-Fenton reagent, N,N'-bis(2-hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthaldiimide (NP-III), induce apoptosis in HL-60 (human promyelocytic leukemia) cells involving the activation of caspase-3.