Observer-based deadbeat predictive speed controller for surface-mounted PM synchronous motor.

This paper investigates an observer-based deadbeat predictive speed controller (DBPSC) for a prototype surface-mounted permanent magnet synchronous motor (SPMSM) drive system. Unlike the conventional deadbeat controllers which are mainly used for deadbeat current control in the inner loop in a cascaded control structure, the proposed deadbeat controller is designed by incorporating a speed controller with a load torque disturbance observer. Specifically the proposed deadbeat speed control law consists of a deadbeat stabilizing feedback control term and a compensation control term. The stability of the proposed closed-loop system is assured based on the former term by forcing all poles of the dynamic error vector toward the origin within a short transient time, which guarantees a fast transient response of the control system by using the solution of an optimal control problem. The latter term is proposed based on the predicted q-axis current to compensate for the model nonlinearities and parameter uncertainties. Next, the deadbeat load torque observer (DBLTO) is proposed to provide a precisely estimated load torque with a high estimation rate for the predicted q-axis current in the latter term, which helps the proposed scheme in obtaining a small steady-state error (SSE), a low total harmonic distortion (THD), and robustness against the parameter variations. In addition, the proposed scheme possesses a simple structure, so it reduces the computational burden for digital implementation. The Lyapunov theory is utilized in the stability proof process of the proposed observer-based method. Finally, the comparative studies between the proposed DBPSC and a conventional deadbeat speed controller (DBSC) are performed under the sudden changes of speed and load torque through simulations on MATLAB/Simulink and experiments on a prototype SPMSM drive using a TI TMS320F28335 DSP platform.

The presence of monoglucosylated N196-glycan is important for the structural stability of storage protein, arylphorin.

Although N-glycosylation has been known to increase the stability of glycoproteins, it is difficult to assess the structural importance of glycans in the stabilization of glycoproteins. APA (Antheraea pernyi arylphorin) is an insect hexamerin that has two N-glycosylations at Asn196 and Asn344 respectively. The glycosylation of Asn344 is critical for the folding process; however, glycosylation of Asn196 is not. Interestingly, the N196-glycan (glycosylation of Asn196) remains in an immature form (Glc1Man9GlcNAc2). The mutation of Asn196 to glutamine does not change the ecdysone-binding activity relative to that of the wild-type. In the present study, we determined the crystal structure of APA, and all sugar moieties of the N196-glycan were clearly observed in the electron-density map. Although the sugar moieties of the glycan generally have high structural flexibility, most sugar moieties of the N196-glycan were well organized in the deep cleft of the subunit interface and mediated many inter- and intrasubunit hydrogen bonds. Analytical ultracentrifugation and GdmCl (guanidinium chloride) unfolding experiments revealed that the presence of the N196-glycan was important for stabilizing the hexameric state and overall stability of APA respectively. Our results could provide a structural basis for studying not only other glycoproteins that carry an immature N-glycan, but also the structural role of N-glycans that are located in the deep cleft of a protein.

Comparative genomic organization of the human and bovine PRNP locus.

We sequenced a 208-kb BAC clone spanning the bovine prion protein (PRNP) locus, and compared the genomic structure with that of human. As a result, we determined the precise breakpoint between the two syntenic genomes, located on the 5' UTR of the PRNP gene, and discovered two highly repetitive sequences near the breakpoint. Further analysis demonstrated that the genomic structure of three genes, PRNP, PRND, and RASSF2, within the syntenic region of the bovine genome is highly conserved in order and orientation. The PRNT locus was not found in bovine but is conserved in several primates, including human. Moreover, we confirmed that the bovine RASSF2 is composed of 10 exons, as is the human gene, showing some difference from a previous report. Our findings may provide useful clues for understanding the evolutional process in the PRNP locus and also the mechanism that allows TSE from cattle to infect humans.

Comparative analysis of chimpanzee and human Y chromosomes unveils complex evolutionary pathway.

The mammalian Y chromosome has unique characteristics compared with the autosomes or X chromosomes. Here we report the finished sequence of the chimpanzee Y chromosome (PTRY), including 271 kb of the Y-specific pseudoautosomal region 1 and 12.7 Mb of the male-specific region of the Y chromosome. Greater sequence divergence between the human Y chromosome (HSAY) and PTRY (1.78%) than between their respective whole genomes (1.23%) confirmed the accelerated evolutionary rate of the Y chromosome. Each of the 19 PTRY protein-coding genes analyzed had at least one nonsynonymous substitution, and 11 genes had higher nonsynonymous substitution rates than synonymous ones, suggesting relaxation of selective constraint, positive selection or both. We also identified lineage-specific changes, including deletion of a 200-kb fragment from the pericentromeric region of HSAY, expansion of young Alu families in HSAY and accumulation of young L1 elements and long terminal repeat retrotransposons in PTRY. Reconstruction of the common ancestral Y chromosome reflects the dynamic changes in our genomes in the 5-6 million years since speciation.

Identification of novel allele on the locus 47z (DXYS5) in the Korean population.

Two homogenous sequences of 47z (DXYS5) are located on the X (DXYS5X) and Y (DXYS5Y) chromosomes, and these are known to be useful polymorphic markers for tracing male-specific gene flow such as the migration routes of human populations. Using the 47z/StuI PCR-RFLP system, we found a novel allele which showed two bands, in contrast to the previous two allele types, one band (Y1) and three bands (Y2). This means that copies of PCR products derived from both the DXYS5X and DXYS5Y loci were clearly cut by the StuI enzyme, implying that the DXYS5X locus of the X chromosome is polymorphic. Allelic frequencies examined in 267 male Korean individuals showed that 95.8% had Y1, 3.4% Y2, and 0.8% had the novel allele. Our findings should contribute to a better understanding of genetic polymorphism on X and Y chromosomes, the molecular evolution mechanism of sex chromosomes, and how the migration route of Koreans is related to those of other East Asian populations.

Oligomerization and multimerization are critical for angiopoietin-1 to bind and phosphorylate Tie2.

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation; however, little is known about the structure-function relationships of Ang1 with its receptor, Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2'). In this study, we generated several Ang1 and angiopoietin-2 (Ang2) variants to define the role of the superclustering and oligomerization domains of the Ang1 protein. Then we analyzed the molecular structure of the variants with SDS-PAGE and rotary metal-shadowing transmission electron microscopy (RMSTEM) and determined the effects of these variants on the binding and activation of Tie2. Ang1 exists as heterogeneous multimers with basic trimeric, tetrameric, and pentameric oligomers, whereas Ang2 exists as trimeric, tetrameric, and pentameric oligomers. The variant Ang1C265S, consisting of trimers, tetramers, and pentamers without multimeric forms of Ang1, yielded less Tie2 activation than did Ang1, whereas monomeric Ang1 (Ang1/FD), dimeric Ang1 variants (Ang1D2, and Ang1D3), and dimeric and trimeric Ang1 variant (Ang1D1) dramatically lost their ability to bind and activate Tie2. An Ang1 protein in which two cysteines (amino acids 41 and 54) were replaced with serines (Ang1C41S/C54S) formed mostly dimers and trimers that were not able to bind and activate Tie2. In addition, improper creation of a new cysteine in Ang2 (Ang2S263C) dramatically induced Ang2 aggregation without activating Tie2. In conclusion, proper oligomerization of Ang1 having at least four subunits by the intermolecular disulfide linkage involving cysteines 41 and 54 is critical for Tie2 binding and activation. Thus, our data shed a light on the structure-function relationships of Ang1 with Tie2.

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells.

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.

Biological characterization of angiopoietin-3 and angiopoietin-4.

The angiopoietin (Ang) family of growth factors includes Ang1, Ang2, Ang3, and Ang4, all of which bind to the endothelial receptor tyrosine kinase Tie2. Ang3 (mouse) and Ang4 (human) are interspecies orthologs. In experiments with human endothelial cell lines, Ang3 was identified as an antagonist of Tie2 and Ang4 was identified as an agonist of Tie2. However, the biological roles of Ang3 and Ang4 are unknown. We examined the biological effect of recombinant Ang3 and Ang4 proteins in primary cultured endothelial cells and in vivo in mice. Recombinant Ang3 and Ang4 formed disulfide-linked dimers. Ang4 (400 ng/mL) markedly increased Tie2 and Akt phosphorylation in primary cultured HUVECs whereas Ang3 (400 ng/mL) did not produce significant changes. Accordingly, Ang4, but not Ang3, induced survival and migration in primary cultured HUVECs. Unexpectedly, intravenously administered Ang3 (30 microg) was more potent than Ang4 (30 microg) in phosphorylating the Tie2 receptor in lung tissue from mice in vivo. Accordingly, Ang3 was more potent than Ang4 in phosphorylating Akt in primary cultured mouse lung microvascular endothelial cells. Ang3 and Ang4 both produced potent corneal angiogenesis extending from the limbus across the mouse cornea in vivo. Thus, Ang3 and Ang4 are agonists of Tie2, but mouse Ang3 has strong activity only on endothelial cells of its own species.

Designed angiopoietin-1 variant, COMP-Ang1, protects against radiation-induced endothelial cell apoptosis.

Radiation therapy is a widely used cancer treatment, but it causes side effects even when localized radiotherapy is used. Extensive apoptosis of microvascular endothelial cells of the lamina propria is the primary lesion initiating intestinal radiation damage after abdominal radiation therapy. Many in vitro studies suggest that angiopoietin-1 (Ang1) has potential therapeutic applications in enhancing endothelial cell survival. For in vivo use, we developed a soluble, stable, and potent Ang1 variant, COMP-Ang1. COMP-Ang1 is more potent than native Ang1 in phosphorylating the Tie2 receptor in lung endothelial cells in vivo. Interestingly, COMP-Ang1 administered i.v. was mainly localized to microvascular endothelial cells of the intestinal villi and lung but not to microvascular endothelial cells of the liver. In irradiated mice, i.v. COMP-Ang1 protected against radiation-induced apoptosis in microcapillary endothelial cells of the intestinal villi and prolonged survival. Thus, COMP-Ang1 could be used as a therapeutic protein for specific protection against endothelial cell injury.

COMP-Ang1: a designed angiopoietin-1 variant with nonleaky angiogenic activity.

Angiopoietin-1 (Ang1) has potential therapeutic applications in inducing angiogenesis, enhancing endothelial cell survival, and preventing vascular leakage. However, production of Ang1 is hindered by aggregation and insolubility resulting from disulfide-linked higher-order structures. Here, by replacing the N-terminal portion of Ang1 with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP), we have generated a soluble, stable, and potent Ang1 variant, COMP-Ang1. This variant is more potent than native Ang1 in phosphorylating the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor and Akt in primary cultured endothelial cells, enhancing angiogenesis in vitro and increasing adult angiogenesis in vivo. Thus, COMP-Ang1 is an effective alternative to native Ang1 for therapeutic angiogenesis in vivo.