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Urinary tract diseases are common in cats, and often require surgical reconstruction. Here, to explore the possibility of urinary tract reconstruction in cats using in-body tissue architecture (iBTA), biosheets fabricated using iBTA technology were implanted into the feline bladder and the regeneration process was histologically evaluated. The biosheets were prepared by embedding molds into the dorsal subcutaneous pouches of six cats for 2 months. A section of the bladder wall was removed, and the biosheets were sutured to the excision site. After 1 and 3 months of implantation, the biosheets were harvested and evaluated histologically. Implantable biosheets were formed with a success rate of 67%. There were no major complications following implantation, including tissue rejection, severe inflammation, or infection. Urinary incontinence was also not observed. Histological evaluation revealed the bladder lumen was almost entirely covered by urothelium after 1 month, with myofibroblast infiltration into the biosheets. After 3 months, the urothelium became multilayered, and mature myocytes and nerve fibers were observed at the implantation site. In conclusion, this study showed that tissue reconstruction using iBTA can be applied to cats, and that biosheets have the potential to be useful in both the structural and functional regeneration of the feline urinary tract.

Cryptic sites are binding pockets that are transiently formed in an apo form or that are induced by ligand binding. The investigation of cryptic sites is crucial for drug discovery, since these sites are ubiquitous in disease-related human proteins, and targeting them expands the number of drug targets greatly. However, although many computational studies have attempted to identify cryptic sites, the detection remains challenging. Here, we aimed to characterize and detect cryptic sites in terms of structural fluctuations in an apo form, investigating proteins each of which possesses a cryptic site. From their X-ray structures, we saw that aromatic residues tended to be found in cryptic sites. To examine structural fluctuations of the apo forms, we performed molecular dynamics (MD) simulations, producing probability distributions of the solvent-accessible surface area per aromatic residue. To detect aromatic residues in cryptic sites, we have proposed a "cryptic-site index" based on the distribution, demonstrating the performance via several measures, such as recall and specificity. Besides, we found that high-ranking aromatic residues were likely to probe concaves in a cryptic site. This implies that such fluctuations provide a profile of scaffolds of compounds with the potential to bind to a particular cryptic site.

Membrane permeability is an important property of drugs in adsorption. Many prediction methods work well for small molecules, but the prediction of middle-molecule permeability is still difficult. In the present study, we modified a classical permeability model based on Fick's law to study passive membrane permeability. The model consisted of the distribution of solute from water to membrane and the diffusion of solute in each solvent. The diffusion coefficient is the inverse of the resistance, and we examined the inertial resistance in addition to the viscous resistance, the latter of which has been widely used in permeability prediction. Also, we examined three models changing the balance between the diffusion of solute in membrane and the conformational change of solute. The inertial resistance improved the prediction results in addition to the viscous resistance. The models worked well not only for small molecules but also for middle molecules, whose structures have more conformational freedom., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.

Bipolar disorder is a major mental illness characterized by severe swings in mood and activity levels which occur with variable amplitude and frequency. Attempts have been made to identify mood states and biological features associated with mood changes to compensate for current clinical diagnosis, which is mainly based on patients' subjective reports. Here, we used infradian (a cycle > 24 h) cyclic locomotor activity in a mouse model useful for the study of bipolar disorder as a proxy for mood changes. We show that metabolome patterns in peripheral blood could retrospectively predict the locomotor activity levels. We longitudinally monitored locomotor activity in the home cage, and subsequently collected peripheral blood and performed metabolomic analyses. We then constructed cross-validated linear regression models based on blood metabolome patterns to predict locomotor activity levels of individual mice. Our analysis revealed a significant correlation between actual and predicted activity levels, indicative of successful predictions. Pathway analysis of metabolites used for successful predictions showed enrichment in mitochondria metabolism-related terms, such as "Warburg effect" and "citric acid cycle." In addition, we found that peripheral blood metabolome patterns predicted expression levels of genes implicated in bipolar disorder in the hippocampus, a brain region responsible for mood regulation, suggesting that the brain-periphery axis is related to mood-change-associated behaviors. Our results may serve as a basis for predicting individual mood states through blood metabolomics in bipolar disorder and other mood disorders and may provide potential insight into systemic metabolic activity in relation to mood changes.

Mitochondria are key players in aging and in the pathogenesis of age-related diseases. Recent mitochondrial transcriptome analyses revealed the existence of multiple small mRNAs transcribed from mitochondrial DNA (mtDNA). Humanin (HN), a peptide encoded in the mtDNA 16S ribosomal RNA region, is a neuroprotective factor. An in silico search revealed six additional peptides in the same region of mtDNA as humanin; we named these peptides small humanin-like peptides (SHLPs). We identified the functional roles for these peptides and the potential mechanisms of action. The SHLPs differed in their ability to regulate cell viability in vitro. We focused on SHLP2 and SHLP3 because they shared similar protective effects with HN. Specifically, they significantly reduced apoptosis and the generation of reactive oxygen species, and improved mitochondrial metabolism in vitro. SHLP2 and SHLP3 also enhanced 3T3-L1 pre-adipocyte differentiation. Systemic hyperinsulinemic-euglycemic clamp studies showed that intracerebrally infused SHLP2 increased glucose uptake and suppressed hepatic glucose production, suggesting that it functions as an insulin sensitizer both peripherally and centrally. Similar to HN, the levels of circulating SHLP2 were found to decrease with age. These results suggest that mitochondria play critical roles in metabolism and survival through the synthesis of mitochondrial peptides, and provide new insights into mitochondrial biology with relevance to aging and human biology.

Bipolar disorder, also known as manic-depressive illness, causes swings in mood and activity levels at irregular intervals. Such changes are difficult to predict, and their molecular basis remains unknown. Here, we use infradian (longer than a day) cyclic activity levels in αCaMKII (Camk2a) mutant mice as a proxy for such mood-associated changes. We report that gene-expression patterns in the hippocampal dentate gyrus could retrospectively predict whether the mice were in a state of high or low locomotor activity (LA). Expression of a subset of circadian genes, as well as levels of cAMP and pCREB, possible upstream regulators of circadian genes, were correlated with LA states, suggesting that the intrinsic molecular circuitry changes concomitant with infradian oscillatory LA. Taken together, these findings shed light onto the molecular basis of how irregular biological rhythms and behavior are controlled by the brain., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Schnurri-2 (Shn-2), an nuclear factor-κB site-binding protein, tightly binds to the enhancers of major histocompatibility complex class I genes and inflammatory cytokines, which have been shown to harbor common variant single-nucleotide polymorphisms associated with schizophrenia. Although genes related to immunity are implicated in schizophrenia, there has been no study showing that their mutation or knockout (KO) results in schizophrenia. Here, we show that Shn-2 KO mice have behavioral abnormalities that resemble those of schizophrenics. The mutant brain demonstrated multiple schizophrenia-related phenotypes, including transcriptome/proteome changes similar to those of postmortem schizophrenia patients, decreased parvalbumin and GAD67 levels, increased theta power on electroencephalograms, and a thinner cortex. Dentate gyrus granule cells failed to mature in mutants, a previously proposed endophenotype of schizophrenia. Shn-2 KO mice also exhibited mild chronic inflammation of the brain, as evidenced by increased inflammation markers (including GFAP and NADH/NADPH oxidase p22 phox), and genome-wide gene expression patterns similar to various inflammatory conditions. Chronic administration of anti-inflammatory drugs reduced hippocampal GFAP expression, and reversed deficits in working memory and nest-building behaviors in Shn-2 KO mice. These results suggest that genetically induced changes in immune system can be a predisposing factor in schizophrenia.

Background: Synaptosomal-associated protein, 25 kDa (SNAP-25) regulates the exocytosis of neurotransmitters. Growing evidence suggests that SNAP-25 is involved in neuropsychiatric disorders, such as schizophrenia, attention-deficit/hyperactivity disorder, and epilepsy. Recently, increases in anxiety-related behaviors and epilepsy have been observed in SNAP-25 knock-in (KI) mice, which have a single amino acid substitution of Ala for Ser187. However, the molecular and cellular mechanisms underlying the abnormalities in this mutant remain unknown., Results: In this study, we found that a significant number of dentate gyrus (DG) granule cells was histologically and electrophysiologically similar to immature DG neurons in the dentate gyrus of the adult mutants, a phenomenon termed the "immature DG" (iDG). SNAP-25 KI mice and other mice possessing the iDG phenotype, i.e., alpha-calcium/calmodulin-dependent protein kinase II heterozygous mice, Schnurri-2 knockout mice, and mice treated with the antidepressant fluoxetine, showed similar molecular expression patterns, with over 100 genes similarly altered. A working memory deficit was also identified in mutant mice during a spontaneous forced alternation task using a modified T-maze, a behavioral task known to be dependent on hippocampal function. Chronic treatments with the antiepileptic drug valproate abolished the iDG phenotype and the working memory deficit in mutants., Conclusions: These findings suggest that the substitution of Ala for Ser187 in SNAP-25 induces the iDG phenotype, which can also be caused by epilepsy, and led to a severe working memory deficit. In addition, the iDG phenotype in adulthood is likely an endophenotype for at least a part of some common psychiatric disorders.

Influenza viruses have developed resistance to current drugs, creating a need for new antiviral targets and new drugs to treat influenza virus infections. In this study, computational and experimental screening of an extensive compound library identified THC19, which was able to suppress influenza virus replication. This compound had no cytotoxic effects and did not disrupt cell cycle progression or induce apoptosis in MDCK cells as confirmed by WST-1 assays, flow cytometry analysis, and caspase-3 assays. Time-of-addition experiments showed that THC19 acts at a relatively early stage of the viral lifecycle. Subsequent mini-genome assays revealed that THC19 inhibited viral genome replication and/or transcription, suggesting that it interferes with one or more of the viral components that form the ribonucleoprotein complexes, namely polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA), nucleoprotein (NP) and viral RNA. Finally, mini-genome assays where PB2, PB1, PA or NP from A/WSN/33 (H1N1) virus were replaced with those from A/Udorn/307/1972 (H3N2) virus effectively demonstrated that THC19 inhibited viral multiplication in a manner dependent upon the PA subunit. Taken together, these results suggest that influenza virus PA protein is a potential target for, and may aid the development of, novel compounds that inhibit influenza A virus replication., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The prion protein (PrP) resides in lipid rafts in vivo, and lipids modulate misfolding of the protein to infectious isoforms. Here we demonstrate that binding of recombinant PrP to model raft membranes requires the presence of ganglioside GM1. A combination of liquid- and solid-state NMR revealed the binding sites of PrP to the saccharide head group of GM1. The binding epitope for GM1 was mapped to the folded C-terminal domain of PrP, and docking simulations identified key residues in the C-terminal region of helix C and the loop between strand S2 and helix B. Crucially, this region of PrP is linked to prion resistance in vivo, and structural changes caused by lipid binding in this region may explain the requirement for lipids in the generation of infectious prions in vitro., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aims: Humanin (HN) is a 24-amino acid peptide that has been shown to have an anti-apoptotic function against neuronal cell death caused by Alzheimer's disease. Increased oxidative stress, one of the major factors contributing to this cell death, also plays an important role in the inflammatory process of atherosclerosis. The current study was designed to test the hypothesis that HN is expressed in the human vascular wall and may protect against oxidative stress., Methods and Results: HN expression in the vascular wall was detected by immunostaining in the endothelial cell layer of human internal mammary arteries (n = 5), atherosclerotic coronary arteries (n = 17), and sections of the greater saphenous vein (n = 3). HN mRNA was expressed in the human aortic endothelial cells (HAECs). Cytoprotective effects of HN against oxidative stress were tested in vitro in HAECs. Pre-treatment with 0.1 µM HN reduced oxidized LDL (Ox-LDL)-induced (i) formation of reactive oxygen species by 50%, (ii) apoptosis by ∼50% as determined by TUNEL staining, and (iii) formation of ceramide, a lipid second messenger involved in the apoptosis signalling cascade, by ∼20%., Conclusion: The current study demonstrates for the first time the expression of HN in the endothelial cell layer of human blood vessels. Exogenous addition of HN to endothelial cell cultures was shown to be effective against Ox-LDL-induced apoptosis. These findings suggest that HN may play a role and may have a protective effect in early atherosclerosis in humans.

The generation of T cell receptor (TCR) sequence diversity can produce 'forbidden' clones able to recognize self-antigens. Here, the structure of the complex between a myelin basic protein peptide (MBP85-99), human leukocyte antigen (HLA)-DR2 (DRB1*1501/DRA) and TCR-Ob.2F3, the dominant autoimmune clone obtained from a multiple sclerosis (MS) patient, has been determined using structural docking simulation and dynamics in silico and compared to the structure of TCR-Ob.1A12 complexes with the same MHC/peptide determined by X-ray crystallography. The two TCRs differ by three amino acids in the CDR3 α and β loops. As the result different hydrogen bonds are formed between the two CDR3β loops and the peptide in the complexes of the simulated structures, with three hydrogen bonds seen in the TCR-Ob.2F3 complex and five in the TCR-Ob.1A12 complex. The two TCRs, each located near the N-terminal end of the HLA-DR2 binding groove and both had an orthogonal binding axis but they deviated by about 10°. Simulation methods, such as structural docking and molecular dynamics as used here, provide an avenue to understand molecular binding mode efficiently and more rapidly than obtaining multiple crystal structures when a large structural database is already available., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Thermal stability of p53 is crucial in preventing cancer proliferation. Critical mutations which significantly destabilize p53 conformation prevent normal interaction between p53 and DNA and consequently interfere with its inhibitory function against cancer proliferation. The purpose of this study was to discover the small compounds called 'chemical chaperons' that can efficiently stabilize the functional p53 conformation and restore the anti-cancer activity. To search for such compounds, we performed a docking simulation using the AutoDock program and the ZINC database. Simply based on the docking energy, we extracted 70 compounds (GJC1-GJC70) and examined their anti-cancer activity using the MTT assay of the human colon cancer cells, HCT116. We found that two compounds, GJC29 and GJC30, significantly inhibited the proliferation of cancer cells compared to the positive control staurosporine. Interaction between p53 and novel anti-cancer compounds were confirmed using SPR measurements. Intriguingly, in the simulated binding mode, both compounds bind to the pocket in the vicinity of the residue V143, one of the mutation hot-spots in p53. Finally, we injected each compound subcutaneously into the nude mice implanted with HCT116 and found that GJC29 has a strong suppressive effect against cancer proliferation in vivo. In conclusion, p53 is an appropriate target for the rational design of the chemical chaperon for cancer treatment.

Transmissible spongiform encephalopathies are associated with the conformational conversion of the prion protein from the cellular form (PrP(C)) to the scrapie form. This process could be disrupted by stabilizing the PrP(C) conformation, using a specific ligand identified as a chemical chaperone. To discover such compounds, we employed an in silico screen that was based on the nuclear magnetic resonance structure of PrP(C). In combination, we performed ex vivo screening using the Fukuoka-1 strain-infected neuronal mouse cell line at a compound concentration of 10 microM and surface plasmon resonance. Initially, we selected 590 compounds according to the calculated docked energy and finally discovered 24 efficient antiprion compounds, whose chemical structures are quite diverse. Surface plasmon resonance studies showed that the binding affinities of compounds for PrP(C) roughly correlated with the compounds' antiprion activities, indicating that the identification of chemical chaperones that bind to the PrP(C) structure and stabilize it is one efficient strategy for antiprion drug discovery. However, some compounds possessed antiprion activities with low affinities for PrP(C), indicating a mechanism involving additional modulation factors. We classified the compounds roughly into five categories: (i) binding and effective, (ii) low binding and effective, (iii) binding and not effective, (iv) low binding and not effective, and (v) acceleration. In conclusion, we found a spectrum of compounds, many of which are able to modulate the pathogenic conversion reaction. The appropriate categorization of these diverse compounds would facilitate antiprion drug discovery and help to elucidate the pathogenic conversion mechanism.

Since the first determination of structure of the HLA-A2 complex, >200 MHC/peptide structures have been recorded, whereas the available T cell receptor (TCR)/peptide/MHC complex structures now are <20. Among these structures, only six are TCR/peptide/MHC Class II (MHCII) structures. The most recent of these structures, obtained by using TCR-Ob.1A12 from a multiple sclerosis patient and the MBP85-99/HLA-DR2 complex, was very unusual in that the TCR was located near the N-terminal end of the peptide-binding cleft of the MHCII protein and had an orthogonal angle on the peptide/MHC complex. The unusual structure suggested the possibility of a disturbance of its signaling capability that could be related to autoimmunity. Here, homology modeling and a new simulation method developed for TCR/peptide/MHC docking have been used to examine the positioning of the complex of two additional TCRs obtained from the same patient (TCR-Ob.2F3 or TCR-Ob.3D1 with MBP85-99/HLA-DR2). The structures obtained by this simulation are compatible with available data on peptide specificity of the TCR epitope. All three TCRs from patient Ob including that from the previously determined crystal structure show a counterclockwise rotation. Two of them are located near the N terminus of the peptide-binding cleft, whereas the third is near the center. These data are compatible with the hypothesis that the rotation of the TCRs may alter the downstream signaling.

To construct a new model of the propagation mechanism of infectious scrapie-type prion protein (PrP(Sc)), here we conducted a disruption simulation of a PrP(Sc) nonamer using structure-based molecular dynamics simulation method based on a hypothetical PrP(Sc) model structure. The simulation results showed that the nonamer disrupted in cooperative manners into monomers via two significant intermediate states: (1) a nonamer with a partially unfolded surface trimer and (2) a hexamer and three monomers. Dimers and trimers were rarely observed. Then, we propose a new PrP(Sc) propagation mechanism where a hexamer plays an essential role as a minimum infectious unit.

A continuous-wave probed laser-induced temperature jump system was constructed and applied to monitor the changes in tryptophan fluorescence of the beta-lactoglobulin during its folding; the kinetic phases were traced from 300 ns to 10 ms after a temperature jump. Notably, an early phase with typical squeezed-exponential characteristics, [exp[-(kt)(beta)], beta>1.0], was observed around several tens of microseconds after the temperature jump, which is actually the earliest phase ever observed for beta-lactoglobulin. This process can be explained by conformational shift occurring within the unfolded ensemble (U-->U'), which is followed by the non-native intermediate (I) formation of this protein.

Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrP(C)) to the scrapie form (PrP(Sc)) is still unknown. Here we discovered a chemical chaperone to stabilize the PrP(C) conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a "pocket" created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrP(Sc). Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrP(C), will open the way to the development of novel anti-prion drugs.

Biomechanical properties of the bone-titanium interface have rarely been studied, due to the technical limitations involved; whether biological bonding mechanisms exist has not been determined. We hypothesized that a selected set of proteoglycan/glycosaminoglycan complexes plays a role in establishing the adhesion between bone and titanium, and utilized the rat bone-marrow-derived osteoblastic culture model to gain an insight into the hypothesis. Gene expression of selected proteoglycan core proteins was up-regulated in the osteoblasts cultured on titanium compared with those on polystyrene. Various sulfated glycosaminoglycans were immunochemically localized at mineralized tissue-titanium interfaces. The administration of various glycosaminoglycan-degrading enzymes into the cultures resulted in a 25-45% reduction of the tissue-titanium interfacial strength, measured by a nanoscratch test; while the hardness and elastic modulus of the mineralized tissue, evaluated by nano-indentation, were not altered. In conclusion, glycosaminoglycan degradation resulted in a decreased interfacial strength between cultured mineralized tissue and titanium, but did not alter the intrinsic strength of the mineralized tissue, suggesting a role for proteoglycan/glycosaminoglycan complexes in the establishment of tissue-titanium adhesion.

The src SH3 domain has been known to be a two-state folder near room temperature. However, in a previous study with an all-atom model simulation near room temperature, the transition state of this protein was not successfully detected on a free-energy profile using two axes: the radius of gyration (R(g)) and native contact reproduction ratio (Q value). In this study, we focused on an atom packing effect to characterize the transition state and tried another analysis to detect it. To explore the atom packing effect more efficiently, we introduced a charge-neutralized all-atom model, where all of the atoms in the protein and water molecules were treated explicitly, but their partial atomic charges were set to zero. Ten molecular dynamics simulations were performed starting from the native structure at 300 K, where the simulation length of each run was 90 ns, and the protein unfolded in all runs. The integrated trajectories (10 x 90 = 900 ns) were analyzed by a principal component analysis (PCA) and showed a clear free-energy barrier between folded- and unfolded-state conformational clusters in a conformational space generated by PCA. There were segments that largely deformed when the conformation passed through the free-energy barrier. These segments correlated well with the structural core regions characterized by large phi-values, and the atom-packing changes correlated with the conformational deformations. Interestingly, using the same simulation data, no significant barrier was found in a free-energy profile using the R(g) and Q values for the coordinate axes. These results suggest that the atom packing effect may be one of the most important determinants of the transition state., ((c) 2006 Wiley-Liss, Inc.)

Unlabelled: This study revealed that osteoblasts generate harder, stiffer, and more delamination-resistant mineralized tissue on titanium than on the tissue culture polystyrene, associated with modulated gene expression, uniform mineralization, well-crystallized interfacial calcium-phosphate layer, and intensive collagen deposition. Knowledge of this titanium-induced alteration of osteogenic potential leading to enhanced intrinsic biomechanical properties of mineralized tissue provides novel opportunities and implications for understanding and improving bone-titanium integration and engineering physiomechanically tolerant bone., Introduction: Bone-titanium integration is a biological phenomenon characterized by continuous generation and preservation of peri-implant bone and serves as endosseous anchors against endogenous and exogenous loading, of which mechanisms are poorly understood. This study determines the intrinsic biomechanical properties and interfacial strength of cultured mineralized tissue on titanium and characterizes the tissue structure as possible contributing factors in biomechanical modulation., Materials and Methods: Rat bone marrow-derived osteoblastic cells were cultured either on a tissue culture-grade polystyrene dish or titanium-coated polystyrene dish having comparable surface topography. Nano-indentation and nano-scratch tests were undertaken on mineralized tissues cultured for 28 days to evaluate its hardness, elastic modulus, and critical load (force required to delaminate tissue). Gene expression was analyzed using RT-PCR. The tissue structural properties were examined by scanning electron microscopy (SEM), collagen colorimetry and localization with Sirius red stain, mineral quantification, and localization with von Kossa stain and transmission electron microscopy (TEM)., Results: Hardness and elastic modulus of mineralized tissue on titanium were three and two times greater, respectively, than those on the polystyrene. Three times greater force was required to delaminate the tissue on titanium than that on the polystyrene. SEM of the polystyrene culture displayed a porous structure consisting of fibrous and globular components, whereas the titanium tissue culture appeared to be uniformly solid. Cell proliferation was remarkably reduced on titanium. Microscopic observations revealed that the mineralized tissue on titanium was composed of uniform collagen-supported mineralization from the titanium interface to the outer surface, with intensive collagen deposition at tissue-titanium interface. In contrast, tissue on the polystyrene was characterized by collagen-deficient mineralization at the polystyrene interface and calcium-free collagenous matrix formation in the outer tissue area. Such characteristic microstructure of titanium-associated tissue was corresponded with upregulated gene expression of collagen I and III, osteopontin, and osteocalcin mRNA. Cross-sectional TEM revealed the apposition of a high-contrast and well-crystallized calcium phosphate layer at the titanium interface but not at the polystyrene interface., Conclusions: Culturing osteoblasts on titanium, compared with polystyrene, enhances the hardness, elastic modulus, and interfacial strength of mineralized tissue to a higher degree. Titanium per se possesses an ability to alter cellular phenotypes and tissue micro- and ultrastructure that result in enhanced intrinsic biomechanical properties of mineralized tissue.

There is a controversy about the major determinants of the folding rate of small single-domain proteins. To shed light on this issue, we examined a possibility that the major determinants may change depending on temperature by conducting molecular dynamics simulations for 17 small single-domain proteins using an off-lattice Go-like model over a wide range of temperature. It was shown that the rank order of the folding rates is temperature dependent, which indicates that the major determinants are dependent on temperature. It was also found that as temperature is decreased, the correlation of the folding rate with the chain length becomes weakened, whereas that with the native topology becomes enhanced. Our simulation results, therefore, may provide a clue to reconcile the apparent controversy between the study by Plaxco based on experimental data and the previous theoretical and subsequent simulation studies: the former showed that the folding rate of two-state folders does not correlate with the chain length but correlates well with the native topology, whereas the latter showed that the folding rate does correlate with the chain length. We propose a possible scenario reconciling the controversy, explaining the reason why the correlation of the folding rate with the chain length became weakened and that with the native topology became enhanced with decreasing temperature.

The biological mechanisms underlying bone-titanium integration and biomechanical properties of the integrated bone are poorly understood. This study assesses intrinsic biomechanical properties of mineralized tissue cultured on titanium having different surface topographies. The osteoblastic phenotypes associated with mineral deposition and collagen synthesis underlying the biomechanical modulation are also reported. Rat bone marrow-derived osteoblastic cells were cultured either on the machined titanium disc or acid-etched titanium disc. Nano-indentation study of day 28 culture revealed that the mineralized tissue on the acid-etched surface shows 3-3.5 times greater hardness than that on the machined surface (p < 0.01). Elastic modulus of the mineralized tissue was also 2.5-3 times greater on the acid-etched surface than on the machined surface (p < 0.01). After 28 days of culture, mineralized nodule area was significantly lower on the acid-etched surface than on the machined surface (p = 0.0105), while total calcium deposition did not differ between the two surfaces, indicating denser mineral deposition on the acid-etched surface. Osteopontin and osteocalcin gene expressions assayed by the reverse transcriptase-polymerase chain reaction were upregulated in the acid-etched titanium culture. Collagen synthesis measured by Sirius red stain-based colorimetry was 1.5-10 times higher on the acid-etched surface than on the machined surface in the initial culture period of day 1 to day 14 (p < 0.0001). The amount of collagen synthesis corresponded with the enhanced gene expression of prolyl 4-hydroxylase, a key enzyme for post-translational modification of collagen chains. Scanning electron microscopic images revealed that tissue cultured on the acid-etched titanium exhibited plate-like, compact surface morphology, while the tissue on the machined titanium appeared porous and was covered by fibrous and punctate structures. We conclude that culturing osteoblasts on rougher titanium surfaces enhances hardness and elastic modulus of the mineralized tissue, associated with condensed mineralization, accelerated collagen synthesis, and upregulated expression of selected bone-related genes.

We previously studied the so-called strange kinetics in the two-dimensional lattice HP model. To further study the strange kinetics, folding processes of a 27-mer cubic lattice protein model with Gō potential were investigated by simulating how the bundle of folding trajectories, consisting of a number of independent Monte Carlo simulations, evolves as the folding reaction proceeds, covering a wide range of temperature. Three realms of folding kinetics were observed depending on temperature. Although at temperatures where folding was two-state-like, the kinetics was conventional single exponential, we found that the time course data were well represented by a squeezed (or "shrunken") exponential function, exp [-(t/tau)beta] with beta > 1, at temperatures lower than the folding temperature, where folding was fastest and of a nonglassy downhill type. The squeezed exponential kinetics was found to pertain to the subdiffusion on the nonglassy downhill free energy surface and presents a marked contrast both to the single exponential kinetics and to the stretched exponential kinetics that was observed at lower temperatures where folding was also downhill but topological frustration came into effect. The observed temperature dependence of the folding kinetics suggests that some small single-domain proteins may follow the squeezed exponential kinetics at about the room temperature., (Copyright 2004 Wiley-Liss, Inc.)

A simple lattice model of protein folding is studied in order to analyze the kinetic partitioning phenomena in the energy landscape perspective. By restricting the area of conformational space, it becomes possible to follow many Monte Carlo trajectories until they reach equilibrium. Alteration of population of trajectories is monitored and the relations between the energy landscape and kinetics are examined. Kinetic partitioning phenomena are categorized into different types in terms of characteristic time constants and partitioning ratio. In a specific partitioning process, refolding proceeds along the parallel pathways; the time constants have a temperature dependence similar to that observed in hen lysozyme. High-energy conformations are classified into groups according to the probability that the trajectories starting from those conformations will reach each energy valley. The partitioning ratio is determined by the way in which the conformational space is organized into these groups., (Copyright 2001 Wiley-Liss, Inc.)

We have developed the long-term life support system that enables the experiment of aquatic animals breeding for 90 days or more for the future experiments in orbit. In order to enable long-term breeding of wide aquatic animals, it is necessary to remove nitrate produced by biological nitrification. Then, we examined a denitrification method to use an electrochemical reaction of biofilm-electrode reactor. In this research, we have not kept the aquatic animals actually but imitated breeding of five goldfish. The ammonia of about 250 ppm was added in breeding water through 90 days. As a result, neither ammonia nor nitrite accumulated 0.1 ppm and nitrate could be suppressed to about 10 ppm.

In STS-95 Space Shuttle mission, an aquatic animal research facility, Vestibular Function Experiment Unit (VFEU), was flown to perform neurobiological experiment with marine fish, oyster toadfish. For this purpose, we have developed a sea water purification system using highly active nitrifying bacteria at low temperature. With this system, the water quality in the VFEU was maintained in sufficient condition to keep the toadfish in healthy state for 9 days of the mission. This report summarizes the efficiency of the filter system based on the results from pre-flight bacterial preparation, water analysis of samples taken during flight, and the post-flight analysis of the bacterial filter.

Vestibular Function Experiment Unit (VFEU), one of the Spacelab facility flown in Neurolab mission (STS-90) in April, 1998, was to support neurophysiological research using a marine fish, Opsanus tau (oyster toadfish). The functions of the VFEU were primarily a quality management of environmental water during the mission at 14 degrees C and for acquiring physiological signals from implanted micro-electrodes in the otolith nerves as well as the spatial acceleration of the fish. A key element of the life support system was a balanced biological filter containing two types of nitrifying bacteria, Nitrosomonas for ammonia oxidization and Nitrobacter for nitrite oxidization. Although the 16 days mission was successful, two toadfishes died in late phase of the mission. Ammonium concentration in those two life support systems elevated to remarkably high level at the end of the mission whereas the other two indicated very low. This report summarizes the results of the water quality management of the VFEU during the Neurolab mission based on analysis of water samples taken during the flight and those taken prior and just after the flight.

We have developed a water purification system that enables long-term experiment with aquatic animals for 90 days or more on the space station. We designed the system that combined a biofilter for ammonia removal (nitrification) with another for nitrate removal (denitrification). The experiment with goldfish was for 90 days with an aquatic animals' examination device. The equipment consists of a fish tank, a filter module, pumps, and an artificial lung for gas exchange. The goldfish were kept in the tank without any water replacement throughout the experiment. When a filter module consists of adsorbents without bacteria, the concentration of the nitrite and ammonia begin to increase so that the goldfish die. On the contrary, neither ammonia nor nitrite accumulated throughout the experiment, and the concentration of T-N also maintained 30 ppm or less when the combined biofilter was used. Moreover, no fish died throughout the period. The water purification system with biofilter enabled us to examine the long-term life support testing. We also report a new denitrification (correction of dentrification) method for the life support system.