Biomedical subjects

Shinsaku Nakagawa

Publications and source records attributed to Shinsaku Nakagawa.

At least 19 recordsLinked to original sources

PubMed2007

Design of a practical fluorescent probe for superoxide based on protection-deprotection chemistry of fluoresceins with benzenesulfonyl protecting groups.

A strategy for designing probes based on protection-deprotection chemistry involving fluoresceins and their benzenesulfonyl (BES) derivatives has led to the development of a much more practical superoxide (O(2) (-.)) probe than the previously reported bis(2,4-dinitro-BES) tetrafluorofluorescein (6 a). Examination of various BES derivatives, developed from the starting point of the prototype probe 6 a, yielded 4,5-dimethoxy-2-nitro-BES tetrafluorofluorescein (BESSo; 7 j) as the optimal reagent. A microtiter plate assay with BESSo showed a tenfold improved detection limit for O(2) (-.) compared with such an assay based on 6 a. BESSo showed markedly better specificity for O(2) (-.) than for GSH or other reactive oxygen species, and this specificity was significantly higher than that of Fe(2+) and some reducing enzymes. These features have resulted in the development of an assay based on BESSo that is capable of providing more unambiguous results for O(2) (-.) release from neutrophils, with or without stimulation by phorbol myristate acetate, as compared with an assay based on 6 a. Intracellular generation of O(2) (-.) in human Jurkat T cells stimulated by butyric acid has been measured by using flow cytometry and fluorescence microscopy utilizing the acetoxymethyl derivative of BESSo.

Cells, Cultured↗

PubMed2006

A strategy for efficient cross-presentation of CTL-epitope peptides leading to enhanced induction of in vivo tumor immunity.

The activation of antitumor cytotoxic T-lymphocytes (CTLs) depends on how efficiently the relevant tumor antigen peptides are delivered into the major histocompatibility complex (MHC) class I presentation pathway in antigen presenting cells (APCs). An elegant approach to promote the peptide-MHC class I association has been described for enhanced peptide transportation into the endoplasmic reticulum (ER) by adding an ER insertion signal sequence (Eriss). Nevertheless, this approach does not appear potent enough to induce in vivo tumor protective immunity. Herein, we present a novel peptide-vaccine strategy based on the combined utilization of Eriss and fusogenic liposomes (FLs) capable of directly introducing encapsulated CTL-epitope peptides into the MHC class I pathway of APCs. APCs pulsed with free peptides, FL-encapsulated peptides, or FL-encapsulated Eriss-conjugated peptides exhibited comparable levels of antigen-presenting activity at early phases after pulsing. Interestingly, whereas in the first two methods the APC ability began to decline 40 to 60 h after pulsing, FL-encapsulated Eriss(+) peptides allowed APCs to retain peptide-presentation activity for at least 140 h. This advantage of FL-encapsulated Eriss(+) peptides correlated with the induction of more potent antitumor immunity compared with soluble Eriss(+) or Eriss(-) peptides or FL-encapsulated Eriss(-) peptides when they were administered in vivo. Thus, Eriss-conjugated CTL-epitope peptides encapsulated in FLs provide a highly efficient tumor-vaccine to enhance the induction of in vivo tumor immunity.

Amino Acid Sequence↗

PubMed2006

Synthesis of 15,20-triamide analogue with polar substituent on the phenyl ring of arenastatin A, an extremely potent cytotoxic spongean depsipeptide.

In order to increase metabolic stability and water solubility of arenastatin A, an extremely potent cytotoxic depsipeptide from the Okinawan marine sponge of Dysidea arenaria, several 15,20-triamide analogues with a polar substituent on the phenyl ring were synthesized. The 15,20-triamide analogues with a polar substituent (24, 30, and 31) showed increased solubility to MeOH and stronger cytotoxicity against KB cells in comparison with the parental 15,20-triamide analogue (2). Furthermore, the diethylamine analogue (30) exhibited in vivo anti-tumor activity against subcutaneously implanted murine sarcoma.

Amides↗

PubMed2006

A novel method for construction of gene fragment library to searching epitopes.

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.

Amino Acid Sequence↗

PubMed2006

Bastadin 6, a spongean brominated tyrosine derivative, inhibits tumor angiogenesis by inducing selective apoptosis to endothelial cells.

Bastadin 6, a macrocyclic tetramer of a brominated tyrosine derivative, was isolated from a marine sponge and its anti-angiogenic activity was evaluated. Bastadin 6 was found to inhibit vascular endothelial growth factor (VEGF)- or basic fibroblast growth factor (bFGF)-dependent proliferation (IC50=0.052 micromol/l) of human umbilical vein endothelial cells (HUVECs) 20- to 100-fold selectively in comparison with normal fibroblast (3Y1) or several tumor cells (KB3-1, K562 and Neuro2A). Bastadin 6 also inhibited VEGF- or bFGF-induced tubular formation (0.1 micromol/l, 6 h treatment) and VEGF-induced migration (1 micromol/l, 4 h treatment) of HUVECs. Moreover, bastadin 6 almost completely blocked VEGF- or bFGF-induced in vivo neovascularization in the mice corneal assay and suppressed growth of s.c. inoculated A431 solid tumor in nude mice (100 mg/kg, i.p.). Bastadin 6 induced cell death of HUVECs with an apoptotic phenotype, whereas it showed no effect on the VEGF-induced auto-phosphorylation of VEGF receptors Flt-1 and KDR/Flk-1. These results suggest that the anti-angiogenic effect of bastadin 6 is closely related to selective induction activity of apoptosis against endothelial cells.

Angiogenesis Inhibitors↗

PubMed2006

Vaccine efficacy of fusogenic liposomes containing tumor cell-lysate against murine B16BL6 melanoma.

Recent advances in tumor immunology have facilitated the development of cancer immunotherapy targeting tumor-associated antigens (TAAs). However, because TAAs were identified in only a few types of human cancer, novel vaccine strategies that utilize tumor cell-lysate (TCL), including unidentified TAAs as an antigen source, are needed. Herein, we describe the utility of fusogenic liposomes (FLs) as TCL-delivery carriers for both ex vivo dendritic cell-based vaccination and in vivo direct immunization in the murine B16BL6 melanoma model. As a result, both in vivo direct immunization and ex vivo immunization induced anti-B16 melanoma prophylactic effects. Ex vivo dendritic cell (DC)-mediated vaccination strategy exert more potent anti-tumor effect than direct immunization. Our results suggest that this flexible system is a promising approach for the development of versatile cancer immunotherapy regimes.

Animals↗

PubMed2006

Non-methylated CpG motif packaged into fusogenic liposomes enhance antigen-specific immunity in mice.

DNA rich in non-methylated CG motifs (CpGs) enhances induction of immune responses against co-administered antigen encoding genes. CpGs are therefore among the promising adjuvants known to date. However, naked plasmid DNA, even which contains CpG motifs, are taken up by antigen presenting cells via the endocytosis pathway. Endocytosed DNAs are thus degraded and their gene expression levels are inefficient. In this context, an effective plasmid delivery carrier is required for DNA vaccine development. We show in the present study that packaging plasmids containing CpGs into fusogenic liposomes (FL) derived from conventional liposomes and Sendai virus-derived active accessory proteins is an attractive method for enhancing the efficacy of a DNA vaccine. These CpG-enhanced plasmids (possessing 16 CpG repeats) that were packaged into FL, enhanced ovalbumin (OVA)-specific T cell proliferation and cytotoxic T cell activity after immunization. In fact, vaccination with CpG enhanced plasmid-loaded FL induced effective prophylactic effects compared with 13 repeats CpG containing plasmid in a tumor challenge experiment. Thus, the development of a CpG-enhanced DNA-FL genetic immunization system represents a promising tool for developing candidate vaccines against some of the more difficult infectious, parasitic, and oncologic disease targets.

Animals↗

PubMed2006

Quality enhancement of the non-immune phage scFv library to isolate effective antibodies.

The non-immune phage antibody library system is one of the most attractive technologies available to current therapeutic, diagnostic and basic scientific research. This system allows the rapid isolation of antibodies of interest that could subsequently be applied directly to drug delivery systems and antibody therapy. Previously, we reported the primer sets to encompass the antibody repertoire and thus improve library quality. However, a wide number of varying primer sets cause to decrease the amplification efficiency of antibody genes. In the present study, we re-generated the library primer sets newly and constructed an improved library from non-immune mice that was far superior in terms of variety and quality. This new library contained 2.4 billion independent clones. In addition, we optimized the selection step from this library to isolate high-affinity antibodies. The optimization of an affinity panning protocol by the incorporation of an automated Microfluidics instrument led to the successful isolation of three different monoclonal antibodies for human vascular endothelial growth factor receptor 2 (KDR). These antibodies were demonstrated to exhibit high specificity and were able to detect a mere 0.6 fmol of KDR by dot blot analysis. Previously reported antibodies for luciferase were also isolated successfully from this library. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies and the resulting isolation of antibodies for clinical and research applications.

Amino Acid Sequence↗

PubMed2006

Creation of novel Protein Transduction Domain (PTD) mutants by a phage display-based high-throughput screening system.

Significant research effort is currently focused on Protein Transduction Domains (PTDs) as potential intracellular drug delivery carriers. However, the application of this technology is limited because the transduction efficiencies are often insufficient for therapeutic purposes, even using HIV-1 Tat peptide. Here we describe a high-throughput screening method based on a phage display system for isolating novel PTDs with improved cell penetration activity. The screening method involves using protein synthesis inhibitory factor (PSIF) as cargo of PTD. Using this method, several Tat-PTD mutants of superior cell-penetrating activity were isolated. Interestingly, the amino acid sequence of the PTD mutants contained some characteristic residues, such as proline. Thus, our screening method may prove useful in determining the relationship between protein transduction and amino acid sequence.

Bacteriophages↗

PubMed2006

Promotion of optimized protein therapy by bioconjugation as a polymeric DDS.

In recent years, clinical applications of recombinantly produced bioactive proteins such as cytokines have attracted attention. However, since these recombinant proteins are rather unstable in vivo, their clinical use as therapeutic agents requires frequent administration at a high dosage. This regimen disrupts homeostasis and results in severe side effects. To overcome these problems, bioactive proteins have been conjugated with water-soluble synthetic (WSS) polymeric carriers. Chemical modification of a protein with a WSS polymeric carrier (bioconjugation) regulates tissue distribution, resulting in a selective increase in its desirable therapeutic effects and a decrease in undesirable side effects. Among several drug delivery system (DDS) technologies, bioconjugation has been recognized as one of the most efficient methods for improving therapeutic potency of proteins. However, for further enhancement of the therapeutic potency and safety of conjugated bioactive proteins, more precise regulation of the in vivo behavior of each protein is necessary for selective expression of its therapeutic effect. Therefore, alternative WSS polymeric modifiers in which new functions such as targeting and controlled release of drugs can be added are required for further development of bioconjugated drugs. Recently, we have synthesized a novel polymeric drug carrier, poly(vinylpyrrolidone-co-dimethyl maleic anhydride) [PVD], which was a powerful candidate drug carrier for cancer therapy. In this review, we introduce useful information that enabled us to design polymeric drug carriers and their application for protein therapy.

Antineoplastic Agents↗

PubMed2006

[Development of nanomedicine using intracellular DDS].

In recent years, sustained release and targeting system using nanospheres or microspheres is noticed in systemic pharmacokinetics. However, in the near future, not only "systemic pharmacokinetics" but also "intracellular pharmacokinetics" seems to be important in Drug Delivery System research. In this context, we have tried to develop the novel cytoplasmic nanoparticle (NP) delivery methods using fusogenic liposomes (FL) and protein transduction domain (PTD). In this study, we demonstrated that the FL efficiently delivered the encapsulated NP to the cytoplasm directly in a fusion -dependent. Oligonucleotides attached to NP were gradually released in the cytoplasm after its efficient delivery using FL. Furthermore, we have succeeded in identifying the novel PTD using phage displayed random peptide library. In near future, this novel PTDs are applied to cytoplasmic NP delivery carrier. From these results, we suggested that this technology is very important to control the intracellular pharmacokinetics, and can be also applied to any NP which will be produced by the nanotechnology in the future.

Cytoplasm↗

PubMed2006

[Biological characteristics of an established model of ovarian cancer in mice and its homologous cell lines].

BACKGROUND & OBJECTIVE: There are no specific methods for early diagnosis of ovarian cancer, recurrence prevention and drug-resistance. The experimental mouse model of ovarian cancer could help to reveal the biological and genetic features of ovarian cancer, and provide rational basis for further intervention strategy. This study was to establish a model of ovarian cancer in mice and homologous cell line, and analyze its biological characteristics. METHODS: Ovarian cancer was developed in 8-week-old female F1 (C57BL/6N x C3H/He) mice by a single whole-body neutron irradiation of 2.7 Gy from a (252)Cf source. A metastatic cell line was established through serial subcutaneous transplantation of the primary tumor for 11 generations, and then tumor cells were transferred to in vitro cultivation. These cells were cloned for more than 6 months. The biological characteristics of the tumors and the homologous cell line were determined by cellular and molecular biological techniques. RESULTS: The grafted tumors in mice were successively passaged for 11 generations with a successful inoculation rate of 96% during 23 months. A tumor cell line OV99 isolated from the grafted tumors was established after 6 months and grew steadily. Morphologic characters and ultrastructures of OV99 cells were accorded with those of ovarian cancer epithelia. The chromosomal analysis of OV99 cells revealed aneuploid pattern of 76 chromosomes. Flow cytometry (FCM) and reverse transcription-polymerase chain reaction (RT-PCR) showed same features between OV99 cells and positive control ovarian cancer cell line OVHM, including distribution of cell cycle, rapid growth rate and the expression of P21, P185, P53, proliferating nuclear cell antigen (PCNA) and Cyclin D proteins, and MAGE-1 and MAGE-3 mRNA. CONCLUSION: Establishment of the ovarian carcinoma animal model in mice and OV99, a cell line owns biologic characteristics of ovarian cancer cells, provides experimental materials for further investigation of ovarian carcinoma.

Animals↗

PubMed2005

Design and synthesis of a Tat-related gene transporter: a tool for carrying the adenovirus vector into cells.

A Tat-related peptide, acetyl-Gly-Arg-Arg-Arg-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, designed to transport an Adenovirus vector (Ad) into cells, was synthesized. The synthetic peptide was conjugated to Ad, which potentially can act as an efficient carrier of heterologous genes into cells. The Tat-related peptide was synthesized using the solid phase method and then was coupled to the heterofunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester. The resulting peptide-succinimidohexanoic acid N-hydroxysuccinimide ester was conjugated to Ad containing the luciferase gene. B16BL6 cells infected with the peptide-conjugated Ad luciferase gene construct exhibit a 50-fold greater luciferase activity than B16BL6 cells infected with wild-type Ad containing the luciferase gene.

Adenoviridae↗

PubMed2005

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release.

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

Animals↗

PubMed2005

RNA interference of PPARgamma using fiber-modified adenovirus vector efficiently suppresses preadipocyte-to-adipocyte differentiation in 3T3-L1 cells.

The peroxisome proliferator-activated receptor (PPAR) gamma is regarded as a "master regulator" of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPARgamma in adipose, RNA interference (RNAi) of PPARgamma should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult to transfect by plasmid vectors and viral vectors. We optimized the transduction of both 3T3-L1 preadipocytes and adipocytes by means of fiber-modified adenovirus (Ad) vectors. Among the various vectors tested, polylysine modification of the C-terminal of the fiber knob most markedly improved the transduction efficiency in both 3T3-L1 preadipocytes and adipocytes. Then, we examined whether fiber-modified Ad vectors with polylysine peptides expressing the small interfering RNA (siRNA) for PPARgamma inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes. Oil red O staining and measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity indicated that the vectors effectively suppressed the differentiation of 3T3-L1 preadipocytes to adipocytes. These results suggested that the combination of fiber-modified Ad vectors containing polylysine peptides and RNAi is an effective tool for the study of the biological and physiological mechanism of adipogenesis in adiposity and diabetes using 3T3-L1 models. Ad vector-mediated RNAi for PPARgamma should also be useful to clarify the biological role of the PPARgamma pathway in various tissues in addition to adipose and for therapeutic application to a variety of diseases, including adiposity and diabetes.

3T3-L1 Cells↗

PubMed2005

A single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with Meth-A fibrosarcoma.

Cytokine-encoding viral vectors are considered to be promising in cancer gene immunotherapy. Interleukin 12 (IL-12) has been used widely for anti-tumor treatment, but the administration route and tumor characteristics strongly influence therapeutic efficiency. Meth-A fibrosarcoma has been demonstrated to be insensitive to IL-12 treatment via systemic administration. In the present study, we developed an IL-12-encoding fiber-mutant adenoviral vector (AdRGD-IL-12) that showed enhanced gene transfection efficiency in Meth-A tumor cells, and the production of IL-12 p70 in the culture supernatant from transfected cells was confirmed by ELISA. In therapeutic experiments, a single low-dose (2 x 10(7) plaque-forming units) intratumoral injection of AdRGD-IL-12 elicited pronounced anti-tumor activity and notably prolonged the survival of Meth-A fibrosarcoma-bearing mice. Immunohistochemical staining revealed that the IL-12 vector induced the accumulation of T cells in tumor tissue. Furthermore, intratumoral administration of the vector induced an anti-metastasis effect as well as long-term specific immunity against syngeneic tumor challenge.

Adenoviridae↗

PubMed2005

Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells.

The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.

Adenoviridae↗

PubMed2005

PEGylated adenovirus vectors containing RGD peptides on the tip of PEG show high transduction efficiency and antibody evasion ability.

BACKGROUND: PEGylation of adenovirus vectors (Ads) is an attractive strategy in gene therapy. Although many types of PEGylated Ad (PEG-Ads), which exhibit antibody evasion activity and long plasma half-life, have been developed, their entry into cells has been prevented by steric hindrance by polyethylene glycol (PEG) chains. Likewise, sufficient gene expression for medical treatment could not be achieved. METHODS: A set of PEG-Ads, which have different PEG modification rates, was constructed, and gene expression was evaluated using A549 cells. A novel PEGylated Ad (RGD-PEG-Ad), which contained RGD (Arg-Gly-Asp) peptides on the tip of PEG, was developed. We evaluated gene expression both in Coxsackie-adenovirus receptor (CAR)-positive as well as -negative cells, and in vivo gene expression was also determined. Furthermore, the antibody evasion ability and the specificity of infection exhibited by this RGD-PEG-Ad were also evaluated. RESULTS: Whereas PEG-Ads decreased gene expression in CAR-positive cells, RGD-PEG-Ad enhanced gene expression notably, to a level about 200-fold higher than that of PEG-Ads. Moreover, gene expression of RGD-PEG-Ad was almost equal to that of Ad-RGD, which contains an RGD-motif in the fiber and exhibits among the highest gene expression of CAR-positive and -negative cells. Furthermore, although Ad-RGD gene expression decreased remarkably in the presence of anti-Ad antiserum, RGD-PEG-Ad maintained its activity against antibodies. In vivo experiments also demonstrated that the modification of Ads with RGD-PEG induced efficient gene expression. CONCLUSIONS: In the present study, we demonstrated that a new strategy, which combined integrin-targeting the RGD peptide on the tip of PEG and modified the Ad using this material, could enhance gene expression in both CAR-positive and -negative cells. At the same time, this novel PEGylated Ad maintained strong protective activity against antibodies. This strategy could also be easily modified for developing other vectors using other targeting molecules.

Adenoviridae↗