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The interaction of proteins with hydrophobic ligands in biological membranes is an important research topic in the life sciences. The hydrophobic nature of ligands, especially their lack of water solubility, often makes it difficult to experimentally investigate their interactions with proteins, thus hampering quantitative evaluation based on thermodynamic parameters. The fatty acid-binding proteins, particularly FABP3, discussed in this review can recognize fatty acids, a primary component of membrane lipids, with high affinity. The precise three-dimensional structure of fatty acids and related ligands bound in FABP3 and their interaction with the binding pocket will contribute to the understanding of accurately determining physicochemical factors that cause the expression of affinity between protein surfaces and lipids in biological membranes. During the research of FABP3, we encountered many of the problems that were widely implicated in experiments dealing with hydrophobic ligands. To address these issues, we developed experimental methodologies using X-ray crystallography, calorimetry, and surface plasmon resonance. Using these methods and computational approaches, we have obtained several insights into the interaction of hydrophobic ligands with protein binding sites. Structural and functional studies of FABP potentially lead to a better understanding of the interaction between lipids and proteins, and thus, this protein may provide one of the model systems for investigating substance transport across cell membranes and inner membrane systems.

Ganglioside GM3 in the plasma membranes suppresses cell growth by preventing the autophosphorylation of the epidermal growth factor receptor (EGFR). Biological studies have suggested that GM3 interacts with the transmembrane segment of EGFR. Further biophysical experiments are particularly important for quantitative evaluation of the peptide-glycolipid interplay in bilayer membranes using a simple reconstituted system. To examine these interactions in this way, we synthesized the transmembrane segment of EGFR bearing a nitrobenzoxadiazole fluorophore (NBD-TM) at the N-terminus. The affinity between EGFR and GM3 was evaluated based on Forster resonance energy transfer (FRET) between NBD-TM and ATTO594-labeled GM3 in bilayers where their non-specific interaction due to lateral proximity was subtracted by using NBD-labeled phospholipid. This method for selectively detecting the specific lipid-peptide interactions in model lipid bilayers disclosed that the lateral interaction between GM3 and the transmembrane segment of EGFR plays a certain role in disturbing the formation of active EGFR dimers.

An efficient peptide purification strategy is established, comprising the selective reaction of an N-terminal N-(methoxy)glycine residue of the peptide and isothiocyanato-functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation-prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240-272). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human β-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys10 reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Platelet-rich plasma (PRP) has been widely applied in regenerative therapy due to its high concentration of growth factors. Previous in vitro and in vivo studies have provided evidence supporting the angiogenic activity of PRP. To more directly demonstrate how PRP acts on endothelial cells, we examined the PRP-induced changes in the motility of human umbilical vein endothelial cells by examining the involvement of VEGF. Time-lapse quantitative imaging demonstrated that in the initial phase (∼2 h) of treatment, PRP substantially stimulated cell migration in a wound-healing assay. However, this effect of PRP was not sustained at significant levels beyond the initial phase. The average net distance of cell migration at 10 h was 0.45 ± 0.16 mm and 0.82 ± 0.23 mm in control and PRP-stimulated cells, respectively. This effect was also demonstrated with recombinant human VEGF and was significantly attenuated by a neutralizing anti-VEGF antibody. Immunofluorescent examination of paxillin and actin fibers demonstrated that PRP concomitantly up-regulated focal adhesion and cytoskeletal formation. Western blotting analysis of phosphorylated VEGFR2 demonstrated that PRP mainly stimulated the phosphorylation of immature VEGFR2 in a dose- and time-dependent manner, an action that was completely blocked by the neutralizing antibody. Taken together, these data suggest that PRP acts directly on endothelial cells via the activation of VEGFR2 to transiently up-regulate their motility. Thus, the possibility that PRP desensitizes target endothelial cells for a relatively long period of time after short-term activation should be considered when the controlled release system of PRP components is designed. © 2015 Wiley Periodicals, Inc.

Introduction The protective effects of vascular endothelial growth factor (VEGF)-A 165 b on kidney tissue have been suggested in animal studies. However, the relevance of urinary and circulating VEGF-A 165 b levels in chronic kidney disease patients remains unclear. Therefore, the present study aimed to investigate the urinary and circulating VEGF-A 165 b levels in patients with chronic kidney disease. Methods This observational study enrolled a total of 92 Japanese patients with chronic kidney disease, who had undergone inulin renal clearance measurements for the accurate assessment of measured GFR. Urinary or circulating total VEGF-A and VEGF-A 165 b levels were measured using enzyme-linked immunosorbent assay. Results Urinary VEGF-A 165 b levels were significantly lower in G3a, G3b, and G4 + G5 category patients than in G1 + G2 category patients. Correlation analysis found that serum creatinine levels, serum cystatin C levels, eGFRcre, eGFRcys, and mGFR were weakly but significantly correlated with urinary VEGF-A 165 b levels. Additionally, circulating VEGF-A 165 b levels were significantly higher in G4 + G5 category patients than in G1 + G2 category patients. Conclusion A low urinary VEGF-A 165 b level reflects renal dysfunction in the chronic kidney disease stage, while a high circulating VEGF-A 165 b level cannot be attributed to decreased renal clearance.

Long-chain fatty acids (FAs) with low water solubility require fatty-acid-binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub-Angstrom X-ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart-type FABP (FABP3) preferentially incorporates a U-shaped FA of C10–C18 using a lipid-compatible water cluster, and excludes longer FAs using a chain-length-limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.

Ladder-shaped polycyclic ethers (LSPs) are predicted to interact with membrane proteins; however, the underlying mechanism has not been satisfactorily elucidated. It has been hypothesized that LSPs possess non-specific affinity to α-helical segments of transmembrane proteins. To verify this hypothesis, we constructed a model LSP interaction system in a lipid bilayer. We prepared 5 types of α-helical peptides and reconstituted them in liposomes. The reconstitution and orientation of these peptides in the liposomes were examined using polarized attenuated total reflection infrared (ATR-IR) spectroscopy and gel filtration. The results revealed that 4 peptides were retained in liposomes, and 3 of them formed stable transmembrane structures. The interaction between the LSP and the peptides was investigated using Förster resonance energy transfer (FRET). In the lipid bilayer, the LSP strongly recognized the peptides that possessed aligned hydrogen donating groups with leucine caps. We propose that this leucine-capped 16-amino acid sequence is a potential LPS binding motif.

An atmospheric-pressure plasma (APP) treatment was recently reported to render titanium (Ti) surfaces more suitable for osteoblastic cell proliferation and osteogenesis. However, the mechanism of action remains to be clearly demonstrated. In this study, we focused on cell adhesion and examined the effects of the APP treatment on the initial responses of human prenatal-derived osteoblastic cells incubated on chemically polished commercially pure Ti (CP-cpTi) plates. In the medium containing 1% fetal bovine serum, the initial cell adhesion and the actin polymerization were evaluated by scanning electron microscopy and fluorescence microscopy. The expression of cell adhesion-related molecules and osteoblast markers at the messenger RNA level was assessed by real-time quantitative polymerase chain reaction. Although the cells on the APP-treated CP-cpTi surface developed fewer cytoskeletal actin fibers, they attached with higher affinity and consequently proliferated more actively (1.46-fold over control at 72 h). However, most of the cell adhesion molecule genes were significantly downregulated (from 40 to 85% of control) in the cells incubated on the APP-treated CP-cpTi surface at 24 h. Similarly, the osteoblast marker genes were significantly downregulated (from 49 to 63% of control) at 72 h. However, the osteoblast marker genes were drastically upregulated (from 197 to 296% of control) in these cells by dexamethasone and β-glycerophosphate treatment. These findings suggest that the APP treatment improves the ability of the CP-cpTi surface to support osteoblastic proliferation by enhancing the initial cell adhesion and supports osteoblastic differentiation when immature osteoblasts begin the differentiation process.

The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate., Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.

The positioning and density of leaf stomata are regulated by three secretory peptides, EPIDERMAL PATTERNING FACTOR 1 (EPF1), EPF2 and stomagen. Several lines of published evidence have suggested a regulatory pathway as follows. EPF1 and EPF2 are perceived by receptor complexes consisting of a receptor-like protein, TOO MANY MOUTHS (TMM), and receptor kinases, ERECTA (ER), ERECTA-LIKE (ERL) 1 and ERL2. These receptors activate a mitogen-activated protein (MAP) kinase module. MAP kinases phosphorylate and destabilize the transcription factor SPEECHLESS (SPCH), resulting in a decrease in the number of stomatal lineage cells. Stomagen acts antagonistically to EPF1 and EPF2. However, there is no direct evidence that EPF1 and EPF2 activate or that stomagen inactivates the MAP kinase cascade, through which they might regulate the SPCH level. Experimental modulation of these peptides in Arabidopsis thaliana would change the number of stomatal lineage cells in developing leaves, which in turn would change the expression of SPCH, making the interpretation difficult. Here we reconstructed this signaling pathway in differentiated leaf cells of Nicotiana benthamiana to examine signaling without the confounding effect of cell type change. We show that EPF1 and EPF2 are able to activate the MAP kinase MPK6, and that both EPF1 and EPF2 are able to decrease the SPCH level, whereas stomagen is able to increase it. Our data also suggest that EPF1 can be recognized by TMM together with any ER family receptor kinase, whereas EPF2 can be recognized by TMM together with ERL1 or ERL2, but not by TMM together with ER.

Object This study aimed to clarify changes in segmental instability following a unilateral approach for microendoscopic posterior decompression and muscle-preserving interlaminar decompression compared with traditional procedures and destabilized models. Methods An ex vivo experiment was performed using 30 fresh frozen porcine functional spinal units (FSUs). Each intact specimen was initially tested for flexion-extension, lateral bending, and torsion up to 1.5° using a material testing system at an angular velocity of 0.1°/second under a preload of 70 N. Microendoscopic posterior decompression, muscle-preserving interlaminar decompression, bilateral medial facetectomy, left unilateral total facetectomy, and bilateral total facetectomy were then performed, followed by mechanical testing with the same loading conditions, in 6 randomized FSUs from each group. Stiffness and neutral zone were standardized by dividing the experimental values by the baseline values and were then compared among groups. Results Mean standardized stiffness values for all loading modes tended to decrease in the order of muscle-preserving interlaminar decompression, microendoscopic posterior decompression, bilateral medial facetectomy, left unilateral total facetectomy, and bilateral total facetectomy. In contrast, mean standardized neutral zone values tended to increase in the order of muscle-preserving interlaminar decompression, microendoscopic posterior decompression, bilateral medial facetectomy, left unilateral total facetectomy, and bilateral total facetectomy. In flexion, values for standardized stiffness following microendoscopic posterior decompression and muscle-preserving interlaminar decompression were higher and standardized neutral zone following microendoscopic posterior decompression and muscle-preserving interlaminar decompression were lower than the values following left unilateral total facetectomy and bilateral total facetectomy while there was no significant difference among bilateral medial facetectomy, left unilateral total facetectomy, and bilateral total facetectomy. Values of standardized stiffness and standardized neutral zone in left torsion following microendoscopic posterior decompression, muscle-preserving interlaminar decompression, and bilateral medial facetectomy were equally superior to values of the destabilization models (left unilateral total facetectomy and bilateral total facetectomy). Except for standardized stiffness in left bending, the values of the parameters for each bending tended to be the same as in the other loading modes. Conclusions The present biomechanical study showed that overall stability of the FSUs was maintained following microendoscopic posterior decompression, muscle-preserving interlaminar decompression, and bilateral medial facetectomy compared with the destabilization models of left unilateral total facetectomy or bilateral total facetectomy. Comparison of the postoperative stability following microendoscopic posterior decompression, muscle-preserving interlaminar decompression, and bilateral medial facetectomy revealed that muscle-preserving interlaminar decompression tended to be superior, followed by microendoscopic posterior decompression and bilateral medial facetectomy.

An efficient peptide purification strategy is established, comprising the selective reaction of an N-terminal N-(methoxy)glycine residue of the peptide and isothiocyanato-functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation-prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240-272). Copyright © 2016 European Peptide Society and John WileySons, Ltd.

Recently, brain injury has been mainly analyzed by computer simulation. However, simulation model of brain needed to obtain accurate analysis results has been shown variety of values. Simulation model reproduced deformational behavior of brain with velocity has not been built. We obtained relaxation curve from hemisphere indentation test for cerebral surface using our own experiment setup, and calculated shear modulus and viscosity. We built viscoelastic model of cerebral surface and evaluated effect of velocity, sites, and leptomeninx on mechanical properties. Our experiment showed that short term shear modulus with leptomeninx was approximately 1.4 times higher values than it without leptomeninx, and viscosity value in high speed range was lower than slow speed. We evinced that brain tissue depends on velocity on high speed range, and leptomeninx affects a rigidity of cerebral surface. Additionally, we confirmed that our viscoelastic model is numerically able to reproduce deformational behavior of brain on high speed range, and acquired parameter values for functions of viscoelastic model.

ObjectThe objective of this study was, using a novel intraoperative measurement (IOM) system, to test the hypothesis that an increased facet joint volume is evidence of spinal instability.MethodsIn 29 patients (male/female ratio 13:16; mean age 67.5 years, range 43–80 years)—17 with degenerative spondylolisthesis (DS) of the lumbar spine (Group DS) and 12 with canal stenosis (CS) of the lumbar spine (Group CS)—DICOM (Digital Imaging and Communications in Medicine) data derived from CT scans were transferred to a workstation. A 3D model of facet joint spaces was reconstructed and the average volume of the bilateral facets was calculated. Segmental properties—stiffness, absorption energy (AE), and neutral zone (NZ)—were measured using an IOM system, and values were compared between groups. Linear regression analyses were performed among biomechanical parameters and average volumes.ResultsStiffness and AE did not differ significantly between groups. The NZ was significantly greater in Group DS than in Group CS (p < 0.05) and significantly positively correlated with the average volume (R2= 0.141, p < 0.05). Stiffness tended to negatively correlate with average volume. Absorption energy did not correlate with average volume.ConclusionsBiomechanical analyses using the IOM system verified that an increased facet joint volume is evidence of spinal instability, represented by NZ, in the degenerative lumbar spine.

Twist drills are often used to prepare implant sites for the internal fixation of fractures, and the stability of the fixation, which depends directly on how securely the implants are held, depends on the quality of the bone around the pilot holes after implant site preparation. To optimize the design of surgical drills, this study evaluated the effects of the drill design elements (thinning, and the helix and point angles) on drilling characteristics (thrust force and temperature increase and duration) when drilling bone. We used mature porcine femurs because porcine bone resembles human bone, and measured the thrust force and bone temperature at three points during the drilling test using drills with different designs and a standard surgical drill. We found that types X, N, and R thinning were effective in reducing the thrust force, and a quick helix was effective in reducing temperature increase and duration when drilling cortical bone. However, no obvious relationship was observed between the drill design elements and the drilling characteristics, perhaps because the cutting system of a drill is complex. In the future, creating a more efficient evaluation method to overcome the complexity of the cutting system and examining the relationship between the drill design elements and the drilling characteristics should be carried out to improve the drilling performance of bone.

A knee joint injury is one of the most common injuries in car-pedestrian accidents. For the examination of the knee joint injury tolerance and the determination of the absolute range of knee joint injury criteria, we performed dynamic three-point bending tests on the porcine knee joint using the own drop impact test setup. During the test, an impactor (mass: 21 kg) was launched toward the lateral side of the specimen placed on a three-point bending support. The femoral epiphysis was completely fractured when the drop height was 700 mm. Multiple damages to the medial collateral ligament and other soft tissues such as the posterior cruciate ligament were observed when the drop height was 500 mm. Only the medial collateral ligament was damaged when the drop height was 300 mm. No injury was observed when the drop height was 100 mm. The calculation of the maximum bending moment acting at the knee joint showed that the injury criterion range was from 136 to 320 Nm. This range includes the values of injury criterion in previous studies on the human knee joint. Thus, there is hardly any difference between the injury criteria for the porcine and human knee joints.

Selective inhibition of protein-protein interactions important for cellular processes could lead to the development of new therapies against disease. In the area of cancer, overexpression of the proteins human double minute 2 (HDM2) and its homolog HDMX has been linked to tumor aggressiveness. Both HDM2 and HDMX bind to p53 and prevent cell cycle arrest or apoptosis in damaged cells. Developing a strategy to simultaneously prevent the binding of both HDM2 and HDMX to p53 is an essential feature of inhibitors to restore p53 activity in a number of different cancers. Inhibition of protein-protein interactions with synthetic molecules is an emerging area of research that requires new inhibitors tailored to mimic the types of interfaces between proteins. Our strategy to create inhibitors of protein-protein interactions is to develop a non-natural scaffold that may be used as a starting point to identify important molecular components necessary for inhibition. In this study, we report an N-acylpolyamine (NAPA) scaffold that supports numerous sidechains in a compact atomic arrangement. NAPAs were constructed by a series of reductive aminations between amino acid derivatives followed by acylation at the resulting secondary amine. An optimized NAPA was able to equally inhibit the association of both HDM2 and HDMX with p53. Our results demonstrate some of the challenges associated with targeting multiple protein-protein interactions involved in overlapping cellular processes.

An efficient method of peptide thioester synthesis is described. The reaction is based on an N-4,5-dimethoxy-2-mercaptobenzyl (Dmmb) auxiliary-assisted N-S acyl shift reaction after assembling a peptide chain by Fmoc-solid phase peptide synthesis. The Dmmb-assisted N-S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S-peptide with sodium 2-mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on-resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41-mer peptide thioester, a phosphorylated peptide thioester and a 33-mer peptide thioester containing a trimethylated lysine residue.

In late years, the patient load of chronic respiratory failure disease, such as a Obstructive Pulmonary Disease (COPD) or Sleep Apnea Syndrome (SAS) increaseschronic obstructive pulmonary disease (COPD) and sleep apnea syndrome (SAS), is increasing. For the patient, in whom such condition is comparatively serious, non-invasive positive pressure ventilation (NPPV) may be prescribed. Since this disposal method uses a nasal mask, a patient is treated non-invasively. Although, It is reported that skin obstacles arise, such as a pressure necrosis and compression gangrene, by the reason the mask pressed the face.Consequentially, it was created that the finite element model of the face portion based on a human body slice picture, and the stress which acts on the contact area of a nasal mask was calculated. And, It turned out that a nasal mask suitable for the form of an individual face is required, in order to reduce a skin obstacle.

Here we investigated the biomechanical properties of spinal segments in patients with degenerative lumbar spondylolisthesis (DLS) using a novel intraoperative measurement system. The measurement system comprised spinous process holders, a motion generator, a load cell, an optical displacement transducer, and a computer. Cyclic displacement of the holders produced flexion-extension of the segment with all ligamentous structures intact. Stiffness, absorption energy (AE), and neutral zone (NZ) were determined from the load-deformation data. Forty-one patients with DLS (M/F = 15/26, mean age 68.6 years; Group D) were studied. Adjacent segments with normal discs in six patients (M/F = 3/3, mean age 35 years) were included as a control group (Group N). Flexion stiffness was significantly lower in Group D than in Group N. The NZ, however, was significantly greater in Group D than in Group N. Thus, compared to normal segments, spinal segments with DLS had a lower flexion stiffness and a higher NZ. NZs in Group D were, however, widely distributed compared to those in Group N that showed NZ 2 mm/N can be considered as unstable.