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Biomedical subjects

S Futaki

Publications and source records attributed to S Futaki.

At least 19 recordsLinked to original sources
PubMed2007

Activation of c-Jun NH2-terminal kinase (JNK) pathway during islet transplantation and prevention of islet graft loss by intraportal injection of JNK inhibitor.

AIMS/HYPOTHESIS: Although application of the Edmonton protocol has markedly improved the outcome for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has remained low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. The aim of this study was to map the c-Jun NH2-terminal kinase (JNK) pathway that mediates islet loss during islet transplantation and to clarify whether intraportal injection with JNK inhibitor during islet transplantation can prevent islet graft loss. METHODS: We measured JNK activity in the liver, fat and muscle of diabetic mice and in the liver immediately after islet transplantation. We examined the effect of intraportal injection of JNK inhibitory peptide at islet transplantation. RESULTS: JNK activity became progressively higher at least until 24 h after transplantation. The cell-permeable peptide of JNK inhibitor was delivered not only in the liver but also in other insulin target organs, preventing JNK activation in the liver at least until 24 h after transplantation and reducing JNK activity in these insulin target organs. Moreover, the peptide inhibitor prevented islet graft loss immediately after transplantation and improved islet transplant outcome. CONCLUSIONS/INTERPRETATION: These findings suggest that control of the JNK pathway is extremely important in islet transplantation and that intraportal injection of JNK inhibitor during islet transplantation (addition of JNK inhibitor to transplant media) could prevent the impairment of islet cells, leading to improved outcome for pancreatic islet transplantation.

Adipose Tissue↗
PubMed2005

The transcriptional landscape of the mammalian genome.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

3' Untranslated Regions↗
PubMed2004

Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy.

Since endosomal escape and the nuclear delivery of plasmid DNA (pDNA) constitute major barriers for transgene expression, a quantitative evaluation of intracellular trafficking of pDNA would be highly desirable in terms of optimizing a nonviral gene delivery system. In the present study, a novel strategy is proposed for the quantification of rhodamine-labeled pDNA in endosomes/lysosomes, cytosol, and nucleus. Endosomes/lysosomes and nucleus were stained with LysoSensor DND-189 and Hoechst 33258, respectively, to distinguish them from the cytosol. The pixel areas of the clusters derived from the rhodamine were used as an index for the amount of pDNA. This approach was applied to the analysis of the intracellular trafficking of pDNA transfected by LipofectAMINE PLUS, stearylated octaarginine (STR-R8), and octaarginine (R8). In the case of R8, most of the pDNA was trapped by endosomes/lysosomes. STR-R8 exhibited endosomal escape followed by nuclear translocation in a time-dependent manner. LipofectAMINE PLUS was the most effective in rapidly delivering pDNA to the nucleus as well as the cytosol. These differences in the intracellular trafficking of pDNA correlated well with the transgene expression. Therefore, this method enables the quantitative analysis of the intracellular pharmacokinetics of pDNA and promises to provide useful information for optimizing nonviral gene delivery systems.

Animals↗
PubMed2004

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation.

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

3T3 Cells↗
PubMed2002

Relationship of the fistulas to the rectum and genitourinary tract in mouse fetuses with high anorectal malformations induced by all-trans retinoic acid.

Since high anorectal malformations with fistulae in human embryos and fetuses of successive developmental stages have not been reported, the embryologic relationship between the rectal fistula (RF) and the genitourinary tract (GUT) in high anorectal agenesis (ARA) remains to be elucidated. This study investigates the developmental relationship between the RF and the GUT in male and female fetuses with high ARA using our established model for high ARA with fistula in mice. Pregnant mice received all-trans retinoic acid suspended in corn oil (5 mg/ml) 100 mg/kg i.p. on day 9 of pregnancy. All fetuses were removed from the uterus on a single day from days 12 to 18 of pregnancy. The caudal regions were analyzed histologically with hematoxylin and eosin staining. All fetuses examined had high ARA with fistula. On day 12 of pregnancy, an anomalous communication was seen between the urogenital sinus (UGS) and the rectum. In the affected female fetuses, on day 14 of pregnancy the paramesonephric (müllerian) ducts and müllerian tubercle were located above the rectocloacal fistula (RCF), and on day 18 of pregnancy the uterovaginal canal was located between the cloaca and the RCF. In the male fetuses, on day 14 of pregnancy the junction between the mesonephric (wolffian) duct and the UGS was located away from the junction between the rectum and the UGS. On day 18 of pregnancy the ejaculatory duct was located between the urinary bladder and the rectourethral fistula. The results of our experiment clearly show the embryologic relationship between the RF and the GUT with high ARA. The anomalous communication between the UGS and the rectum may interfere with normal caudal migration along the dorsal wall of the UGS at the junction between the UGS and the mesonephric or paramesonephric duct.

Anal Canal↗
PubMed2001

Alamethicin-leucine zipper hybrid peptide: a prototype for the design of artificial receptors and ion channels.

In this report, we describe a novel concept of extramembrane control of channel peptide assembly and the eventual channel current modulation. Alamethicin is a peptide antibiotic, which usually forms ion channels in various association states. By introducing an extramembrane leucine zipper segment (Alm-LeuZ), the association number of alamethicin was effectively controlled to produce a single predominant channel open state. The assembly was estimated to be a tetramer, by comparison of the channel conductance with that of the template-assembled Alm-LeuZ tetramer, which was prepared by the conjugation of a maleimide-functionalized peptide template with cysteine-derivatized Alm-LeuZ segments. Employment of an extramembrane segment of a random conformation provided higher levels of channel conductance. The result exemplified the possibility of channel current control by a conformational switch of the extramembrane segments.

Alamethicin↗
PubMed2001

A 'cassette' RNase: site-selective cleavage of RNA by RNase S equipped with RNA-recognition segment.

RNase S is a unique protein comprising the non-covalent association of two components, the S-peptide and the S-protein. An RNA-recognition segment derived from the human immunodeficiency virus (HIV)-1 Rev protein was conjugated with the S-peptide to form a complex with the S-protein. The resulting RNase S bearing the RNA-recognition segment preferentially hydrolyzed a single position of the RNA stem-loop derived from the specific binding site for the Rev protein.

Amino Acid Sequence↗
PubMed2001

Stoichiometry of subunit e in rat liver mitochondrial H(+)-ATP synthase and membrane topology of its putative Ca(2+)-dependent regulatory region.

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H(+)-ATP synthase are homologous with the Ca(2+)-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca(2+)-dependent regulation of H(+)-ATP synthase activity. In this study, we determined the content of subunit e in H(+)-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca(2+)-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H(+)-ATP synthase revealed that 1 mol of H(+)-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F(0) is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H(+)-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca(2+)-dependent regulatory region of subunit e is exposed on the surface of the C-side of F(0) and that subunit e is involved in the regulation of mitochondrial H(+)-ATP synthase activity probably via its putative Ca(2+)-dependent regulatory region.

Animals↗
PubMed2001

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: total synthesis of human big gastrin-II and cholecystokinin (CCK)-39.

Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To overcome the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue and two protecting groups, tBu for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J.K-1.mol-1) and enthalpy (110.5 kJ.mol-1) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the SN1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i) a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degree C for an appropriate period of time for the cleavage and deprotection. Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was further applied to the stepwise synthesis of human big gastrin-II (34 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degree C), the ratios of the desulfated peptides were less than 15%, and the pure sulfated peptides were obtained in around 10% yields.

Amino Acid Sequence↗
PubMed2001

Evaluation of two sperm antigens, rSMP-B and YWK-II, as targets for immunocontraception.

To determine whether sperm membrane components, rSMP-B and YWK-II, are suitable candidates as immunocontraceptives in humans, antifertility activities of the antibodies to the peptide fragments, rSMP-229 and rSMP-230 of rSMP-B and YAL-198 of YWK-II, were examined. In a previous report, anti-rSMP-230 antibody was shown to immobilise human sperm and to block human fertilisation, and the antigen (rSMP-230) to interact with antisperm antibodies found in sera of infertile women. Antibody to the second synthetic peptide, rSMP-229, corresponding to a different segment of rSMP-B, mimicked the biological activities of the anti-rSMP-230 antibody. Anti-YAL-198 antibody significantly, although weakly, inhibited human fertilisation. In the murine model, the anti-rSMP-B antibodies blocked in vitro fertilisation of mouse eggs but had no influence on embryo growth. Anti-YAL-198 antibody, however, arrested the growth of zygotes. In conclusion, rSMP-B, a human sperm protein, is a promising candidate in the development of an immunocontraceptive for human application. A second sperm protein, YWK-II, is effective as an antifertility immunogen in experimental animals.

Amyloid beta-Protein Precursor↗
PubMed2001

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: Part II. Total synthesis of human big gastrin-II and its C-terminal glycine-extended peptide (G34-Gly sulfate) by the solid-phase segment condensation approach.

Application of the fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase segment condensation approach to the preparation of sulfated peptides was investigated through the synthesis of human big gastrin-II, a 34-residue sulfated tyrosine [Tyr(SO3H)]-containing peptide. Highly acid-sensitive 2-chlorotrityl resin (Clt resin) was exclusively employed as an anchor-resin for the preparation of the three peptide segments having the C-terminal Pro residue as well as of the Tyr(SO3H)-containing resin-bound segment. By using the PyBOP-mediated coupling protocol [PyBOP=benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphatel, we successively condensed each segment and constructed the 34-residue peptide-resin without any difficulty. The final acid treatment of the fully protected peptide-resin at low temperature (90% aqueous TFA, 0 degree C for 8 h), which can detach a Tyr(SO3H)-containing peptide from the resin and remove the protecting groups concurrently with minimum deterioration of the sulfate, afforded a crude sulfated peptide. After one-step HPLC purification, a highly homogeneous human big gastrin-II was easily obtained in 14% yield from the protected peptide-resin. The sulfate form of the C-terminal glycine-extended gastrin (G34-Gly sulfate), a posttranslational processing intermediate of gastrin-II, was also successfully prepared with the segment condensation approach (11% yield). These results demonstrated the usefulness of the segment condensation protocol for preparing large Tyr(SO3H)-containing peptides.

Gastrins↗
PubMed2000

Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery.

A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48-60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48-60). These included such peptides as the d-amino acid- and arginine-substituted Tat-(48-60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4 degrees C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg)(n) (n = 4-16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n approximately 8) for the efficient translocation.

Amino Acid Sequence↗
PubMed2000

Identification of a novel myosin-Va mutation in an ataxic mutant rat, dilute-opisthotonus.

Mutations of the myosin-Va gene (Myo5a) cause diluted coat color in mice and are occasionally associated with severe neurological disorders. Dilute-opisthotonus (dop) is a spontaneous gene mutation in the rat, and phenotypes of the homozygote (dop/dop) are similar to those of the Myo5a-deficient mouse, suggesting that the mutation resides in the rat Myo5a gene. To elucidate the molecular basis of the dop mutation, we cloned the rat Myo5a cDNA from the wild type and the dop/dop. The wild-type rat Myo5a cDNA contained a 5487-bp ORF and showed higher homology with Myo5a of the other species than Myr6 (Myo5b) in the rat. A 141-bp in-frame deletion was detected in the head region in the dop cDNA. An intragenic rearrangement consisting of a 306-bp inversion associated with 17-bp and 217-bp deletions were identified in the Myo5a gene of the dop genome. This rearrangement involved a 141-bp exon, which was skipped in the dop transcript. The MyoVA protein expression was severely impaired in the dop/dop brain. This is the first report to define the dop mutation as the Myo5a gene abnormality in the rat.

Amino Acid Sequence↗
PubMed2000

Poly-L-glutamine forms cation channels: relevance to the pathogenesis of the polyglutamine diseases.

We report that long-chain poly-L-glutamine forms cation-selective channels when incorporated into artificial planar lipid bilayer membranes. The channel was permeable to alkali cations and H(+) ions and virtually impermeable to anions; the selectivity sequence based on the single-channel conductance was H(+) >> Cs(+) > K(+) > Na(+). The cation channel was characterized by long-lived open states (often lasting for several minutes to tens of minutes) interrupted by brief closings. The appearance of the channel depended critically on the length of polyglutamine chains; ion channels were observed with 40-residue stretches, whereas no significant conductance changes were detected with 29-residue tracts. The channel-forming threshold length of poly-L-glutamine was thus between 29 and 40 residues. A molecular mechanics calculation suggests a mu-helix (. Biophys. J. 69:1130-1141) as a candidate molecular structure of the channel. The channel-forming nature of long-chain poly-L-glutamine may provide a clue to the elucidation of the pathogenetic mechanism of the polyglutamine diseases, a group of inherited neurodegenerative disorders including Huntington's disease.

Cations, Monovalent↗
PubMed2000

Stabilization of a tyrosine O-sulfate residue by a cationic functional group: formation of a conjugate acid-base pair.

Sulfated tyrosine [Tyr(SO3H)]-containing peptides showed characteristic peak patterns in their liquid secondary-ion mass spectrometry (LSIMS) spectra. Protonated molecules were desulfated more easily than their deprotonated counterparts. Therefore, the stabilities of the Tyr(SO3H) residues were well-reflected by peak patterns in their positive-ion spectra. These intrinsic peak patterns were investigated by comparing the behavior of each Tyr(SO3H) residue in acidic solution. As the peptide chain was lengthened and the number of cationic functional groups increased, the peak representing the [MH]+ of a Tyr(SO3H)-containing peptide became more prominent than that representing the desulfated [MH-SO3]+. These alterations in peptide structure also increased the stability of the Tyr(SO3H) residue in acidic solution. Based on the desulfation mechanism of an aryl monosulfate, we predicted that intramolecular cationic functional groups would stabilize Tyr(SO3H) residues by forming conjugate acid-base pairs (or salt bridges) both in the gaseous phase and in acidic solution. In accordance with this theory, Arg residues would take primary responsibility for this self-stabilization within Tyr(SO3H)-containing peptides. Moreover, a long peptide backbone was expected to have a weak protective effect against desulfation of the [MH]+ in the gaseous phase. Tyr(SO3H) residues were also stabilized by adding an external basic peptide containing multiple Arg residues. Formation of such intermolecular acid-base pairs was demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) which detected conjugated peptide ions. The energetically favorable formation of conjugate acid-base pairs prompted by Tyr(SO3H) residues might be a driving force for protein folding and protein-protein interaction.

Amino Acid Sequence↗
PubMed2000

The engineering, structure, and DNA binding properties of a novel His4-type zinc finger peptide.

We have created a novel His4-type zinc finger protein (H4Sp1) engineered by Cys-->His mutations of the Cys2His2-type zinc finger in transcription factor Sp1. The CD and NMR studies reveal that the His4 domain has Zn(II)-dependent folding properties and similar secondary structures to wild-type Cys2His2 domain. The DNA binding experiments demonstrate that H4Sp1 can bind DNA in a specific way. The present artificial peptide H4Sp1 will provide valuable information about the interaction between a metallopeptide and DNA.

Amino Acid Sequence↗
PubMed1999

Identification of the human sperm protein that interacts with sperm-immobilizing antibodies in the sera of infertile women.

OBJECTIVE: To identify the target antigen of sperm-immobilizing antibodies present in the circulation of infertile women. DESIGN: Laboratory research. SETTING: Academic research laboratory. PATIENT(S): Twenty-nine infertile women with sperm-immobilizing antibodies, 22 infertile women with other disorders, and 20 fertile women. INTERVENTION(S): Titers of antibodies to the sperm protein, rSMP-B, were determined by ELISA using as substrate the synthetic peptide segment (rSMP-230) that corresponds with the hydrophilic domain of rSMP-B. Tests for sperm immobilization and zona pellucida penetration were performed using the human IVF system. MAIN OUTCOME MEASURE(S): Human sera with sperm-immobilizing activity were assayed for the presence of antibodies to rSMP-230. Polyclonal antibodies to rSMP-230 were assessed for the same biologic activities as sperm-immobilizing antibodies. RESULT(S): Antibodies to rSMP-230 were detected in 10 (34%) of 29 sera obtained from women with immunologic infertility. In contrast, only one serum sample (2%) from women without sperm-immobilizing activity had a low titer of antibodies to rSMP-230. Polyclonal antibodies to rSMP-230 completely immobilized human sperm in the presence of complement and blocked sperm penetration across the zona pellucida. CONCLUSION(S): The human sperm protein, rSMP-B, probably is the target antigen of sperm-immobilizing antibodies.

Adult↗