Refining Markov state models for conformational dynamics using ensemble-averaged data and time-series trajectories.

A data-driven modeling scheme is proposed for conformational dynamics of biomolecules based on molecular dynamics (MD) simulations and experimental measurements. In this scheme, an initial Markov State Model (MSM) is constructed from MD simulation trajectories, and then, the MSM parameters are refined using experimental measurements through machine learning techniques. The second step can reduce the bias of MD simulation results due to inaccurate force-field parameters. Either time-series trajectories or ensemble-averaged data are available as a training data set in the scheme. Using a coarse-grained model of a dye-labeled polyproline-20, we compare the performance of machine learning estimations from the two types of training data sets. Machine learning from time-series data could provide the equilibrium populations of conformational states as well as their transition probabilities. It estimates hidden conformational states in more robust ways compared to that from ensemble-averaged data although there are limitations in estimating the transition probabilities between minor states. We discuss how to use the machine learning scheme for various experimental measurements including single-molecule time-series trajectories.

Effect of hyaluronan tetrasaccharides on epidermal differentiation in normal human epidermal keratinocytes.

OBJECTIVE: Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation. In addition, HA has been shown to have different biological activities depending on its molecular weight. It has been reported that HA-mediated CD44 activation regulates keratinocyte differentiation. Therefore, the aim of this study was to investigate the influence of HA tetrasaccharides (HA4) on the regulation of keratinocyte differentiation, CD44 gene expression and CD44-phosphorylated protein in human keratinocytes, and compare HA4 with high molecular weight HA. METHODS: Normal human epidermal keratinocytes (NHEKs) were treated at doses of 1 µg mL(-1) HA or HA oligosaccharides (HA4). After treatment, cell viability was checked using an MTT (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each differentiation marker and CD44 mRNA expression was detected by real-time PCR. Each differentiation marker and CD44-phosphorylated protein was assessed by Western blotting. RESULTS: Hyaluronan and HA4 showed no cytotoxicity up to a dose of 1 µg mL(-1) . On day 3 after HA4 treatment, each differentiation marker mRNA and K10 protein level was higher than that of the control. On day 9, late differentiation marker mRNA and protein levels were increased with HA and HA4 treatment. In addition, HA4 treatment increased the expression of CD44 mRNA, CD44-phosphorylated protein and intracellular calcium concentrations. HA4 enhanced keratinocyte differentiation and increased CD44-phosphorylated protein levels. CONCLUSION: HA4 may induce epidermal differentiation through phosphorylation of CD44.

Differential Mitochondrial Adaptation of the Slow and Fast Skeletal Muscles by Endurance Running Exercise in Streptozotocin-Induced Diabetic Mice.

The skeletal muscle is the main organ responsible for insulin action, and glucose disposal and metabolism. Endurance and/or resistance training raises the number of mitochondria in diabetic muscles. The details of these adaptations, including mitochondrial adaptations of the slow and fast muscles in diabetes, are unclear. This study aimed to determine whether exercise training in streptozotocin (STZ)-induced mice leads to differential adaptations in the slow and fast muscles, and improving glucose clearance. Eight-week-old mice were randomly distributed into normal control (CON), diabetes (DM), and diabetes and exercise (DM+Ex) groups. In the DM and DM+Ex groups, mice received a freshly prepared STZ (100 mg/kg) intraperitoneal injection on two consecutive days. Two weeks after the injection, the mice in the groups ran on a treadmill for 60 min at 20 m/min for a week and subsequently at 25 m/min for 5 weeks (5 days/week). The analyses indicated that running training at low speed (25 m/min) enhanced mitochondrial enzyme activity and expression of lactate and glucose transporters in the plantaris (low-oxidative) muscle that improved whole-body glucose metabolism in STZ-induced diabetic mice. There were no differences in glucose transporter expression levels in the soleus (high-oxidative) muscle. The endurance running exercise at 20-25 m/min was sufficient to induce mitochondrial adaptation in the low-oxidative muscles, but not in the high-oxidative muscles, of diabetic mice. In conclusion, the present study indicated that running training at 25 m/min improved glucose metabolism by increasing the mitochondrial enzyme activity and glucose transporter 4 and monocarboxylate transporter 4 protein contents in the low-oxidative muscles in STZ-induced diabetic mice.

Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy.

This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid ß (Aß), the causal protein of Alzheimer's disease. The scFv-transfectant iPS-MP exhibited efficient Aß-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

Plasmin induces degradation and dysfunction of laminin 332 (laminin 5) and impaired assembly of basement membrane at the dermal-epidermal junction.

BACKGROUND: The epidermal basement membrane (BM), located at the dermal-epidermal junction (DEJ), plays important roles not only in adhesion between epidermis and dermis, but also in controlling skin functions. In sun-exposed skin, the BM becomes disrupted and multilayered. In order to explore the impairment of BM assembly, we have used a skin-equivalent (SE) as a model of BM damage and previously clarified the involvement of matrix metalloproteinases (MMPs) in impairment of BM assembly. OBJECTIVES: In this work, we examined the role of urokinase-type plasminogen activator (uPA) and plasmin in impairment of BM assembly at the DEJ by using the SE, as ultraviolet irradiation to the skin increases uPA as well as MMPs. METHODS: SEs were used as a model of formation and damage of BM. Human uPA was detected by enzyme-linked immunosorbent assay and zymography, and gelatinases such as MMP-2 and MMP-9 were detected by zymography. Human plasminogen was added at 0.06 micromol L(-1) (about 3% of plasma level) to increase plasmin to a pathological level. N-terminal peptide sequence analysis of plasmin-treated laminin 332 was carried out to identify alpha3, beta3 and gamma2 chains of laminin 332 and their cleavage sites of each chain. Plasmin-treated laminin 332 was analysed in keratinocyte adhesion activity and binding to type VII collagen. RESULTS: Human uPA was detected in addition to MMP-2 and MMP-9, in conditioned medium of SE. Although the BM was well organized in the presence of an MMP inhibitor alone, the activated plasmin disorganized the BM even in the presence of the inhibitor. The impairment of BM assembly made the epidermis thinner as compared with that of a control cultured in the presence of MMP inhibitor, indicating that the BM affects the polarity and differentiation of the epidermis. The addition of aprotinin, a serine proteinase inhibitor, and tranexamic acid, a uPA-plasmin inhibitor, inhibited the plasmin-induced impairment of BM assembly and facilitated BM reorganization, thereby improving the epidermal structure. N-terminal peptide sequence analysis of plasmin-treated laminin 332 revealed the removal of a 5- or 10-kDa fragment, including the cell adhesion region, from the G3 domain of the alpha3 chain, and the LN domain, which binds to the noncollagenous 1 domain in type VII collagen, from the beta3 chain. Plasmin-treated laminin 332 showed lower keratinocyte adhesion activity and reduced binding to type VII collagen. CONCLUSIONS: These results suggest that uPA and plasmin are involved in the impairment of BM assembly and epidermal differentiation, and that these effects arise at least partly through direct degradation of laminin 332.

Specific reactivity of mild/severe Alzheimer's disease patient's sera to antibody against Abeta1-40 epitope 17-21.

OBJECTIVES: To detect the reactivity pattern of sera from patients with mild and severe Alzheimer's disease (AD) to specific antibodies targeting different epitopes in the primary structure of amyloid-beta (Abeta). MATERIALS AND METHODS: Sera from patients diagnosed with mild or severe AD were used. The reactivity of sera to monoclonal antibodies recognizing 1-7, 5-10, 9-14 and 17-21 epitopes of Abeta1-40 at 36-42 degrees C was determined by an enzyme-linked immunosorbent assay. Proteinase K digestion of Abeta1-40 was investigated by dot blotting at 36 and 40 degrees C. RESULTS: Sera of patients with AD displayed reactivity only with monoclonal antibody recognizing the epitope 17-21 (4G8). The reactivity of sera from patients with severe AD was less than that of sera from patients with mild AD at temperatures 36-41 degrees C, with no difference at 42 degrees C. Patients with severe AD displayed lesser digestion with proteinase K. CONCLUSIONS: Sera derived from patients with AD could react with monoclonal antibodies directed to 17-21 sequences of Abeta1-40 in a temperature-dependent manner. The severity of AD is associated with greater Abeta1-40 aggregation and resistance to proteinase K. The present results may be of value in staging and following up of patients with AD.

Correction: Bottom-up fabrication of artery-mimicking tubular co-cultures in collagen-based microchannel scaffolds.

Correction for 'Bottom-up fabrication of artery-mimicking tubular co-cultures in collagen-based microchannel scaffolds' by A. Tan, et al., Biomater. Sci., 2016, DOI: 10.1039/c6bm00340k.

Bottom-up fabrication of artery-mimicking tubular co-cultures in collagen-based microchannel scaffolds.

We developed a robust bottom-up approach to construct open-ended, tubular co-culture constructs that simulate the human vascular morphology and microenvironment. By design, these three-dimensional artificial vessels mimic the basic architecture of an artery: a collagen-rich extracellular matrix (as the tunica externa), smooth muscle cells (SMCs) (as the tunica media), and an endothelial cell (EC) lining (as the tunica interna). A versatile needle-based fabrication technique was employed to achieve controllable arterial layouts within a PDMS-hosted collagen microchannel scaffold (330 ± 10 µm in diameter): (direct co-culture) a SMC/EC bilayer to follow the structure of an arteriole-like segment; and (encapsulated co-culture) a lateral SMC multilayer covered by an EC monolayer lining to simulate the architecture of a larger artery. Optical and fluorescence microscopy images clearly evidenced the progressive cell elongation and sprouting behavior of SMCs and ECs along the collagen gel contour and within the gel matrix under static co-culture conditions. The progressive cell growth patterns effectively led to the formation of a tubular co-culture with an internal endothelial lining expressing prominent CD31 (cluster of differentiation 31) intercellular junction markers. During a 4-day static maturation period, the artery constructs showed modest alteration in the luminal diameters (i.e. less than 10% changes from the initial measurements). This argues in favor of stable and predictable arterial architecture achieved via the proposed fabrication protocols. Both co-culture models showed a high glucose metabolic rate during the initial proliferation phase, followed by a temporary quiescent (and thus, mature) stage. These proof-of-concept models with a controllable architecture create an important foundation for advanced vessel manipulations such as the integration of relevant physiological functionality or remodeling into a vascular disease-mimicking tissue.

Acotiamide Hydrochloride, a Therapeutic Agent for Functional Dyspepsia, Enhances Acetylcholine-induced Contraction via Inhibition of Acetylcholinesterase Activity in Circular Muscle Strips of Guinea Pig Stomach.

Acotiamide is a first-in-class prokinetic drug approved in Japan for the treatment of functional dyspepsia. Given that acotiamide enhances gastric motility in conscious dogs and rats, we assessed the in vitro effects of this drug on the contraction of guinea pig stomach strips and on acetylcholinesterase (AChE) activity in stomach homogenate following fundus removal. We also investigated the serotonin 5-HT4 receptor agonist mosapride, dopamine D2 receptor and AChE inhibitor itopride, and representative AChE inhibitor neostigmine. Acotiamide (0.3 and 1 µM) and itopride (1 and 3 µM) significantly enhanced the contraction of gastric body strips induced by electrical field stimulation (EFS), but mosapride (1 and 10 µM) did not. Acotiamide and itopride significantly enhanced the contraction of gastric body and antrum strips induced by acetylcholine (ACh), but not that induced by carbachol (CCh). Neostigmine also significantly enhanced the contraction of gastric body strips induced by ACh, but not that by CCh. In contrast, mosapride failed to enhance contractions induced by either ACh or CCh in gastric antrum strips. Acotiamide exerted mixed inhibition of AChE, and the percentage inhibition of acotiamide (100 µM) against AChE activity was markedly reduced after the reaction mixture was dialyzed. In contrast, itopride exerted noncompetitive inhibition on AChE activity. These results indicate that acotiamide enhances ACh-dependent contraction in gastric strips of guinea pigs via the inhibition of AChE activity, and that it exerts mixed and reversible inhibition of AChE derived from guinea pig stomach.

Purification and characterization of a coagulant protein from the venom of Russell's viper.

The coagulant protein from the venom of Russell's viper was purified by means of successive chromatography on Sephadex G-50, DEAE-cellulose and Sephadex G-200. The purified coagulant protein was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight was estimated to be about 100 000 by ultracentrifuge analysis and 130 000 by gel filtration. The coagulant protein contains 11.1% carbohydrate which includes 5.1% hexose (galactose: mannose = 1:1), 5% hexosamine (glucosamine), and 1% neuraminic acid (N-acetylneuraminic acid and N-glycolyneuraminic acid). The isoelectric point is pH 6.3. The results of both sodium dodecyl sulfate electrophoresis and gel filtration in 6 M guanidium chloride suggest that it consists of four polypeptide chains. The coagulant protein functions as an enzyme in activating blood coagulation factor X in the presence of Ca2+. N-a-p-Toluenesulfonyl-L-arginine methyl ester hydrolyzing activity in the preparation definitely decreased during purification and it suggests that the clotting activity is not associated with the esterase activity. The clotting activity is inhibited by diisopropyl phosphorofluoridate and by phenylmethylsulfonyl fluoride, suggesting that the coagulant protein is a serine protease. The optimum pH is between pH 7.0 and pH 8.0. At neutral pH the coagulant protein is stable below 50 degrees C, but is rapidly inactivated above 55 degrees C.

A conformational transition at the N terminus of the prion protein features in formation of the scrapie isoform.

The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27-30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27-30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27-30. Denaturation of PrP 27-30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27-30.

Survey of volume CT dose index in Japan in 2014.

OBJECTIVE: The aims of this study are to propose a new set of Japanese diagnostic reference levels (DRLs) for 2014 and to study the impact of tube voltage and the type of reconstruction algorithm on patient doses. The volume CT dose index (CTDI(vol)) for adult and paediatric patients is assessed and compared with the results of a 2011 national survey and data from other countries. METHODS: Scanning procedures for the head (non-helical and helical), chest and upper abdomen were examined for adults and 5-year-old children. A questionnaire concerning the following items was sent to 3000 facilities: tube voltage, use of reconstruction algorithms and displayed CTDI(vol). RESULTS: The mean CTDI(vol) values for paediatric examinations using voltages ranging from 80 to 100 kV were significantly lower than those for paediatric examinations using 120 kV. For adult examinations, the use of iterative reconstruction algorithms significantly reduced the mean CTDI(vol) values compared with the use of filtered back projection. Paediatric chest and abdominal scans showed slightly higher mean CTDI(vol) values in 2014 than in 2011. The proposed DRLs for adult head and abdominal scans were higher than those reported in other countries. CONCLUSION: The results imply that further optimization of CT examination protocols is required for adult head and abdominal scans as well as paediatric chest and abdominal scans. ADVANCES IN KNOWLEDGE: Low-tube-voltage CT may be useful for reducing radiation doses in paediatric patients. The mean CTDI(vol) values for paediatric scans showed little difference that could be attributed to the choice of reconstruction algorithm.

Immunological significances of invariant chain from the aspect of its structural homology with the cystatin family.

The primary structure of p31 of invariant chain (Ii-chain) shows about 50% homology with those of the cystatin family which are endogenous cysteine protease inhibitors. The binding domains between Ii-chain and HLA-DR-7 were estimated from the structural homology between cystatin and Ii-chain and also between cathepsins and DR-7, respectively. The QL64-71 and GS76-88 of Ii-chain were estimated to be the binding domains with GG45-51 and VS57-63 of HLA-DR7, respectively. The purified human Ii-chain from spleen is capable of forming four molecular forms from monomer to tetramer by redox-potential dependent disulfide bond formation. The Ii-chain inhibits cathepsin L and H competitively as a dimer and the K(i) value for cathepsin L was 4.1 x 10(-8) M, but cathepsin B was not inhibited at all. The Ii-chain showed mainly a dimer (60 kDa) under the assay condition of cathepsins with cysteine and was not degraded by these cathepsins. The Ii-chain may play an important role in the regulation of antigenic peptide presentation to MHC class II.

Participation of cathepsin B in processing of antigen presentation to MHC class II.

Cellular and humoral immune responses to vaccines of hepatitis B type and rabies were inhibited by specific inhibitors of cathepsin B, specific synthetic substrates of cathepsin B and anti-cathepsin B antibody. Therefore the lysosomal cathepsin B of antigen presenting cells plays an essential role in processing of these antigens for presentation to MHC class II. One of the active sites of cathepsin B, VN217-222 shares highly homologous sequences with a part of the desetope, a binding domain of antigenic peptides, VN57-62 of MHC class II, beta-chain. This evidence suggests that the peptides processed by the substrate specificity of cathepsin B exhibit a common affinity to bind with the desetope of MHC class II, beta-chain.

Lysophosphatidylcholine upregulates CD40 ligand expression in newly activated human CD4+ T cells.

Lysophosphatidylcholine (lyso-PC) accumulates in tissues undergoing inflammation and atherosclerosis, where an infiltration of T cells is also seen. We found that lyso-PC increased IFN-gamma production and CD40L expression in CD4+ T cells stimulated with anti-CD3 Ab and recombinant CD80 molecules, whereas lyso-PC did not affect IL-2 and IL-4 production. These results suggest that lyso-PC, in combination with other stimuli, may regulate CD4+ T cell functions to propagate local inflammatory reactions and also imply a novel role played by a modified lipid in the selection of Th1/Th2 immune response as well as in the T cell mediated pathogenesis in atherosclerosis.

Regulation of lymphocyte proliferation by eosinophils via chymotrypsin-like protease activity and adhesion molecule interaction.

We investigated the regulatory mechanisms responsible for release of eosinophil cationic protein (ECP) from eosinophils activated by platelet-activating factor (PAF) and monitored intra-cellular pH (pHi) changes using a pH-sensitive fluorescent probe. We also explored the mechanisms by which eosinophils suppress T-lymphocyte proliferation induced by phytohaemagglutinin (PHA). In these experiments, a separated culture to investigate the ECP-mediated pathway and a coculture to identify the adhesion molecules involved in eosinophil-lymphocyte interactions were employed. Chymostatin (1x10(-6) M) inhibited ECP release by about 50% via stimulation by PAF or recombinant interleukin 5(rIL-5) plus IgG. PAF (1x10(-7) M) raised eosinophil pHi from 6.9 to 7.3 within 20 s and pretreatment of these cells with chymostatin (1x10(-6) M), but not with leupeptin or E64-d, completely prevented this increase. Calcium ionophore A23187 (1x10(-7) M) induced ECP release and raised pHi to within a range similar to that of PAF, however, chymostatin had no effect on either. Chymostatin reversed ECP-mediated suppression of PHA-induced T-lymphocyte proliferation in separated cultures, but not in cocultures. In coculture, eosinophils exhibited the same level of suppression of both CD4(+) and CD8(+) T-cell proliferation in response to PHA. Monoclonal antibodies against CD11a, CD18 and CD54, but not CD11b, restored eosinophil suppression of T-lymphocyte proliferation which was chymostatin-resistant in coculture. Eosinophils were unable to suppress the proliferative response to lymphocytes to anti-CD3 stimulation. In conclusion, chymostatin specifically inhibited both the eosinophil pHi increase and ECP release induced by PAF. Eosinophils regulate PHA-induced T-lymphocyte proliferation via the ECP-mediation associated with chymotrypsin-like protease activity. These cells also control interactions with lymphocyte between adhesion molecules, CD11a, CD18 and CD54.

Enzymatic hydrolysis of haloperidol decanoate and its inhibition by proteins.

When [14C]haloperidol decanoate, a long-acting neuroleptic and an ester of haloperidol and decanoic acid, was incubated in human whole blood and plasma and in rat plasma and homogenates of rat brain, lung, liver, kidney, pancreas and muscle, no hydrolysis of the ester was seen. Although the decanoate was hydrolyzed by partially purified carboxylesterase, addition of rat plasma or liver homogenate to the enzymic reaction mixture resulted in marked inhibition of hydrolysis, whereas addition of the defatted residues of plasma or liver produced only partial inhibition. The enzymic hydrolysis was inhibited also by beta-lipoprotein and albumin, depending on their concentrations. The assumption that interaction between haloperidol decanoate and protein resulted in inhibition of the hydrolytic reaction mediated by the enzyme was validated by kinetic models and experimental data. The kinetics were apparently competitive. Based on the kinetic analysis, the interaction between the decanoate and albumin or beta-lipoprotein was investigated by measuring their equilibrium constants and extent of protein binding. Haloperidol decanoate appeared to interact with several proteins; this was exemplified by other measures of protein binding, an increasing effect of proteins on the solubility, and the partition ratio of the ester. The interaction between haloperidol decanoate and proteins caused marked stabilization of this ester against enzymatic hydrolysis and, thereby, influenced its metabolism.

The effect of augmented atrial hypothermia on atrial refractory period, conduction, and atrial flutter/fibrillation in the canine heart.

The purpose of this study was to test the assumption that the cause for postoperative atrial flutter/fibrillation after cardiopulmonary bypass is inadequate atrial myocardial protection. Dogs were subjected to cardioplegic arrest for 60 minutes without augmented atrial hypothermia (seven dogs, control group) or augmented atrial hypothermia with topical atrial cooling (seven dogs, study group). Twenty-five electrodes (15 on the right atrium and 10 on the left atrium) were fixed on the atria to measure effective refractory period and conduction time. Data were taken before bypass, immediately after bypass, and 2 hours after bypass. During cardioplegic arrest the mean temperatures measured in the atria were significantly lower (p less than 0.001) in the study group (13.5 degrees +/- 7.0 degrees C) than in the control group (23.7 degrees +/- 3.2 degrees C). There was no significant change in the mean effective refractory period after bypass in the control or study groups or in the prevalence of inducibility of atrial flutter/fibrillation by extrastimulation (3/7 dogs in the control group and 2/7 in the study group). During right atrial pacing, total conduction times were significantly longer (p less than 0.025 at cycle lengths of 300 and 350 msec) in the control group (74 +/- 5 msec and 75 +/- 7 msec, respectively) than in the study group (65 +/- 9 msec and 64 +/- 8 msec, respectively) immediately after bypass. Two hours after bypass, however, there were no significant differences under the same conditions between the two groups. There were no significant differences in conduction during left atrial pacing after bypass. Comparing those atria that were inducible with those not inducible demonstrated a significantly increased dispersion of effective refractory period (90 +/- 23 msec versus 74 +/- 18 msec, p less than 0.05) and increased conduction time in the inducible group. We concluded that augmented atrial hypothermia during cardioplegic arrest had no effect on the inducibility of fibrillation, had no effect on repolarization, and had only a small effect on conduction, which resolved within 2 hours after bypass. However, the study demonstrates that when the atria are inducible the substrates are an increased dispersion of refractoriness and a prolongation of conduction time.