Motif-programmed artificial extracellular matrix

Motif-programming is a method for creating artificial proteins by combining functional peptide motifs in a combinatorial manner. This method is particularly well suited for developing liaison molecules that interface between cells and inorganic materials. Here we describe our creation of artificial proteins through the programming of two motifs, a natural cell attachment motif (RGD) and an artificial Ti-binding motif (minTBP-1). The created proteins were found to reversibly bind Ti and to bind MC3T3-E1 osteoblast-like cells. Moreover, although the interaction with Ti was not covalent, the proteins recapitulated several functions of fibronectin, and thus, could serve as an artificial ECM on Ti materials. Because this motif-programming system could be easily extended to create artificial proteins having other biological functions and material specificities, it should be highly useful for application to tissue engineering and regenerative medicine.     

Characterization of a sHsp of Schizosaccharomyces pombe, SpHsp15.8, and the implication of its functional mechanism by comparison with another sHsp, SpHsp16.0

There exist two small heat shock proteins (sHsps) in the fission yeast, Schizosaccharomyces pombe (S. pombe), whose expressions are highly induced by heat stress. We have previously expressed, purified, and characterized one of the sHsps, SpHsp16.0. In this study, we examined the other sHsp, SpHsp15.8. It suppressed the thermal aggregation of citrate synthase (CS) from porcine heart and dithiothreitol-induced aggregation of insulin from bovine pancreas with very high efficiency. Almost one SpHsp15.8 subunit was sufficient to protect one protein molecule from aggregation. Like SpHsp16.0, SpHsp15.8 dissociated into small oligomers and then interacted with denatured substrate proteins. SpHsp16.0 exhibited a clear enthalpy change for denaturation occurring over 60 degrees C in differential scanning calorimetry (DSC). However, we could not observe any significant enthalpy change in the DSC of SpHsp15.8. The difference is likely to be caused by the adhesive characteristics of SpHsp15.8. The oligomer dissociation of SpHsp15.8 and SpHsp16.0 and their interactions with denatured substrate proteins were studied by fluorescence polarization analysis (FPA). Both sHsps exhibited a temperature-dependent decrease of fluorescence polarization, which correlates with the dissociation of large oligomers to small oligomers. The dissociation of the SpHsp15.8 oligomer began at about 35 degrees C and proceeded gradually. On the contrary, the SpHsp16.0 oligomer was stable up to approximately 45 degrees C, but then dissociated into small oligomers abruptly at this temperature. Interestingly, SpHsp16.0 is likely to interact with denatured CS in the dissociated state, while SpHsp15.8 is likely to interact with CS in a large complex. These results suggest that S. pombe utilizes two sHsps that function in different manners, probably to cope with a wide range of temperatures and various denatured proteins.     

Synthesis and biological activities of a pH-dependently activated water-soluble prodrug of a novel hexacyclic camptothecin analog

CH0793076 (1) is a novel hexacyclic camptothecin analog showing potent antitumor activity in various human caner xenograft models. To improve the water solubility of 1, water-soluble prodrugs were designed to generate an active drug 1 nonenzymatically, thus expected to show less interpatient PK variability than CPT-11. Among the prodrugs synthesized, 4c (TP300, hydrochloride) having a glycylsarcosyl ester at the C-20 position of 1 is highly water-soluble (>10 mg/ml), stable below pH 4 and rapidly generates 1 at physiological pH in vitro. The rapid (ca. <1 min) generation of 1 after incubation of TP300 with plasma (mouse, rat, dog and monkey) was also demonstrated. TP300 showed a broader antitumor spectrum and more potent antitumor activity than CPT-11 in various human cancer xenograft models.     

Tissue-dependent preventive effect of metallothionein against DNA damage in dyslipidemic mice under repeated stresses of fasting or restraint

AimsTo investigate the effect of repeated stress on DNA damage in seven organs of dyslipidemic mice, and the preventive role of metallothionein (MT).Main methodsFemale adult 129/Sv wild-type and MT-null mice fed high-fat diet (HFD) were repeatedly subjected to mild stress of fasting or restraint in weeks 2 to 4 of 4-week study period. Serum cholesterol level, DNA damage in the liver, pancreas, spleen, bone marrow, kidney, lung and gastric mucosa, and other parameters were determined.Key findingsBody weights were increased in both types of mice fed HFD compared to those fed standard diet (STD), and further increased by 12 h-fasting, while they were markedly decreased by 1–3 h-restraint. Fasting accelerated accumulation of fat in the liver, and increase in serum cholesterol of both types of mice fed HFD. Feeding of HFD increased DNA damage in the pancreas, spleen and bone marrow of both types of mice, compared with those fed STD. In the wild-type mice fed HFD, 24 h-fasting increased DNA damage in the liver and spleen, while restraint increased the damage in the liver, pancreas, spleen and bone marrow. DNA damage in the cells of organs was markedly increased in the MT-null mice. Specifically, damage in the liver, pancreas, spleen and bone marrow was greatly increased with the intensity of stress increased, and the damage was much greater in the restraint mice than in the fasting mice.SignificanceMT plays a tissue-dependent preventive role against DNA damage in various murine organs induced by repeated stress.     

Gait analysis using gravitational acceleration measured by wearable sensors

A novel method for measuring human gait posture using wearable sensor units is proposed. The sensor units consist of a tri-axial acceleration sensor and three gyro sensors aligned on three axes. The acceleration and angular velocity during walking were measured with seven sensor units worn on the abdomen and the lower limb segments (both thighs, shanks and feet). The three-dimensional positions of each joint are calculated from each segment length and joint angle. Joint angle can be estimated mechanically from the gravitational acceleration along the anterior axis of the segment. However, the acceleration data during walking includes three major components; translational acceleration, gravitational acceleration and external noise. Therefore, an optimization analysis was represented to separate only the gravitational acceleration from the acceleration data. Because the cyclic patterns of acceleration data can be found during constant walking, a FFT analysis was applied to obtain some characteristic frequencies in it. A pattern of gravitational acceleration was assumed using some parts of these characteristic frequencies. Every joint position was calculated from the pattern under the condition of physiological motion range of each joint. An optimized pattern of the gravitational acceleration was selected as a solution of an inverse problem. Gaits of three healthy volunteers were measured by walking for 20 s on a flat floor. As a result, the acceleration data of every segment was measured simultaneously. The characteristic three-dimensional walking could be shown by the expression using a stick figure model. In addition, the trajectories of the knee joint in the horizontal plane could be checked by visual imaging on a PC. Therefore, this method provides important quantitive information for gait diagnosis.     

Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

Introduction

A protein analysis using a mass spectrometry indicated that there are serum proteins showing significant quantitative changes after the administration of infliximab. Among them, connective tissue growth factor (CTGF) seems to be related to the pathogenesis of rheumatoid arthritis (RA). Therefore, this study was conducted to investigate how CTGF is associated with the disease progression of RA.

Methods

Serum samples were collected from RA patients in active or inactive disease stages, and before or after treatments with infliximab. CTGF production was evaluated by ELISA, RT-PCR, indirect immunofluorescence microscopy, and immunoblotting. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, a bone resorption assay and osteoclasts specific catalytic enzymes productions.

Results

The serum concentrations of CTGF in RA were greater than in normal healthy controls and disease controls. Interestingly, those were significantly higher in active RA patients compared to inactive RA patients. Furthermore, the CTGF levels significantly were decreased by infliximab concomitant with the disease amelioration. In addition, tumour necrosis factor (TNF)α can induce the CTGF production from synovial fibroblasts even though TNFα can oppositely inhibit the production of CTGF from chondrocytes. CTGF promoted the induction of the quantitative and qualitative activities of osteoclasts in combination with M-CSF and receptor activator of NF-κB ligand (RANKL). In addition, we newly found integrin αVβ3 on the osteoclasts as a CTGF receptor.

Conclusions

These results indicate that aberrant CTGF production induced by TNFα plays a central role for the abnormal osteoclastic activation in RA patients. Restoration of aberrant CTGF production may contribute to the inhibition of articular destruction in infliximab treatment.     

Isolation of eight microsatellite markers from Moina macrocopa for assessing cryptic genetic structure in the wild

We isolated eight polymorphic microsatellite loci from the zooplankton Moina macrocopa (Straus), which is sensitive to pollutants such as insecticides and heavy metals. The isolated loci were polymorphic, with three to seven alleles among 23 individuals. Expected heterozygosities ranged from 0.167 to 0.787. These loci can be used to examine cryptic genetic structure and to infer the connectivity among metapopulations.     

Mature and juvenile tissue models of regeneration in small fish species

The multitude of cells constituting organisms are fragile and easily damaged day by day. Therefore, maintenance of tissue morphology and function is fundamental for multicellular organisms to attain long life. For proper maintenance of tissue integrity, organisms must have mechanisms that detect the loss of tissue mass, activate the de novo production of cells, and organize those cells into functional tissues. However, these processes are only poorly understood. Here we give an overview of adult and juvenile tissue regeneration models in small fish species, such as zebrafish and medaka, and highlight recent advances at the molecular level. From these advances, we have come to realize that the epidermal and mesenchymal parts of the regenerating fish fin-that is, the wound epidermis and blastema, respectively-comprise heterogeneous populations of cells with different molecular identities that can be termed "compartments." These compartments and their mutual interactions are thought to play important roles in promoting the proper progression of tissue regeneration. We further describe the current understanding of these compartments and discuss the possible approaches to affording a better understanding of their roles and interactions during regeneration.     

Heme-dependent activation of neuronal nitric oxide synthase by cytosol is due to an Hsp70-dependent, thioredoxin-mediated thiol-disulfide interchange in the heme/substrate binding cleft

We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20-30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-μ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination either with the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme.