Wearable Power-Assist Locomotor for Gait Reconstruction in Patients With Spinal Cord Injury: A Retrospective Study.

Wearable robotic exoskeletons (WREs) have been developed from orthoses as assistive devices for gait reconstruction in patients with spinal cord injury. They can solve some problems encountered with orthoses, such as difficulty in independent walking and standing up and high energy consumption during walking. The Wearable Power-Assist Locomotor (WPAL), a WRE, was developed based on a knee-ankle-foot orthosis with a single medial hip joint. The WPAL has been updated seven times during the period from the beginning of its development, in 2005, to 2020. The latest version, launched as a commercialized model in 2016, is available for medical facilities. In this retrospective study, which included updated results from previous reports, all data were extracted from development research records from July 2007 to December 2020. The records were as follows: patient characteristics [the number of participants, injury level, and the American Spinal Injury Association Impairment Scale (AIS) score], the total number of WPAL trials when aggregating the cases with all the versions or only the latest version of the WPAL, and maximum walking performance (functional ambulation category [FAC], distance, and time of continuous walking). Thirty-one patients participated in the development research. The levels of spinal cord injury were cervical (C5-C8), upper thoracic (T3-T6), lower thoracic (T7-T12), and lumbar (L1) in 10, 5, 15, and 1 of the patients, respectively. The numbers of patients with AIS scores of A, B, C, and D were 20, 7, 4, and 0, respectively. The total number of WPAL trials was 1,785, of which 1,009 were used the latest version of the WPAL. Twenty of the patients achieved an FAC score of 4 after an average of 9 (median 8, range 2-22) WPAL trials. The continuous walking distance and time improved with the WPAL were compared to the orthosis. We confirmed that the WPAL improves walking independence in people with a wide range of spinal cord injuries, such as cervical spinal cord injuries. Further refinement of the WPAL will enable its long-term use at home.

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

Effects of rubber elongation factor and small rubber particle protein from rubber-producing plants on lipid metabolism in Saccharomyces cerevisiae.

The common proteins rubber elongation factor (REF) and small rubber particle protein (SRPP) are associated with Hevea brasiliensis (Hb) rubber particles. They are involved in the stability of rubber particles and natural rubber biosynthesis. Recently, we cloned cDNAs encoding REF and SRPP from laticifers of Ficus carica (Fc). In the present study, we overexpressed REF/SRPPs (HbREF, FcREF, and FcSRPP) in Saccharomyces cerevisiae in anticipation of future rubber biosynthesis in recombinant yeast. The proteins were localized in the endoplasmic reticulum and lipid droplets (LDs), and affected LD morphology. Furthermore, their overexpression resulted in an accumulation of neutral lipids and a decrease in yeast cell size. This suggests that REF/SRPPs affect lipid metabolism and lead to a decline in the phospholipid content of yeast. We also found that expression of these proteins induced accumulation of steryl esters and triacylglycerols in yeast. This suggests that the coexpression of REF/SRPPs with key enzymes for the biosynthesis of target lipids in yeast is a promising way of increasing production of important lipids like triacylglycerols and terpenes, and that a protein complex consisting of cis-prenyltransferase (CPT), CPT-like protein, and REF/SRPPs for rubber biosynthesis could be reconstituted on yeast lipid droplets.

Cloning and Aggregation Characterization of Rubber Elongation Factor and Small Rubber Particle Protein from Ficus carica.

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are major latex proteins harvested from Hevea brasiliensis (the rubber tree; HbREF and HbSRPP, respectively). Their amino acid sequences exhibit high homology with each other. In the present study, we cloned two cDNAs encoding REF/SRPP-family proteins (FcREF/SRPP-1 and -2) from the laticifers of Ficus carica (fig tree). The amino acid sequences of these proteins showed high homology not only with each other but also with HbREF and HbSRPP. Recombinant FcREF/SRPP-1 and -2 were expressed in E. coli, and their aggregation properties were examined using a Congo red binding assay, agarose gel electrophoresis, and transmission electron microscopy. FcREF/SRPP-1 formed fibrils when incubated in PBS, and grew to micrometer-sized amorphous aggregates that precipitated rapidly. These aggregation properties of FcREF/SRPP-1 are quite similar to those of HbREF, although the growth rate and size of FcREF/SRPP-1 aggregates were inferior to those of HbREF. FcREF/SRPP-2 also formed aggregates during the incubation, but they did not precipitate, as has been reported for HbSRPP. Our results suggest that FcREF/SRPP-1 and -2 correspond to HbREF and HbSRPP, respectively. These aggregation properties could provide useful benchmarks for classifying REF/SRPP-family proteins as REF or SRPP.

Clinical feasibility of gait training with a robotic exoskeleton (WPAL) in an individual with both incomplete cervical and complete thoracic spinal cord injury: A case study.

BACKGROUND: Patients with tetraplegia can achieve independent gait with lateral-type powered exoskeletons; it is unclear whether medial-type powered exoskeletons allow for this. OBJECTIVE: To investigate gait training with a medial-type powered exoskeleton wearable power-assist locomotor (WPAL) in an individual with incomplete cervical (C5) and complete thoracic (T12) spinal cord injury (SCI). METHODS: The 60-session program was investigated retrospectively using medical records. Upon completion, gait performance was examined using three-dimensional motion analyses and surface electromyography (EMG) of the upper limbs. RESULTS: The subject achieved independent gait with WPAL and a walker in 12 sessions. He continuously extended his right elbow; his left elbow periodically flexed/extended. His pelvic inclination was larger than the trunk inclination during single-leg stance. EMG activity was increased in the left deltoid muscles during ipsilateral foot-contact. The right anterior and medial deltoid muscle EMG activity increased just after foot-off for each leg, as did the right biceps activity. Continuous activity was observed in the left triceps throughout the gait cycle; activity was unclear in the right triceps. CONCLUSIONS: These results suggest the importance of upper limb residual motor function, and may be useful in extending the range of clinical applications for robotic gait rehabilitation in patients with SCI.

Novel internally quenched fluorogenic substrates for angiotensin I-converting enzyme and carboxypeptidase Y.

Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) is one of the most important enzymes in the renin-angiotensin system, a major blood pressure control system in mammals. We synthesized novel internally quenched fluorogenic (IQF) substrates for ACE based on the cleavage site of an angiotensin I, introducing N-methyl anthranic acid (Nma) and N(ε)-2,4-dinitrophenyl-lysine (Lys(Dnp))at the N- and C-terminal regions. Kinetic parameters of the synthesized IQF substrates Nma-Phe-His-Lys(Dnp) and Nma-His-Pro-Phe-Lys(Dnp)-Pro were compared with those of a common peptide substrate for ACE, hippuryl (Hip)-His-Leu. The k(cat)/K(m) values of Nma-Phe-His-Lys(Dnp), Nma-His-Pro-Phe-Lys(Dnp)-Pro, and Hip-His-Leu were 5.12, 1.90, and 0.80 µM(-1) s(-1) for rabbit lung ACE, and 16.0, 7.36, and 0.30 µM(-1) s(-1) for recombinant human (rh)-ACE, respectively. These results indicate that Nma-Phe-His-Lys(Dnp) is an excellent substrate for rh-ACE. Carboxypeptidase Y also hydrolyzed Nma-Phe-His-Lys(Dnp) efficiently with K(m), k(cat), and k(cat)/K(m) values of 60.2 µM, 105 s(-1), and 1.74 µM(-1) s(-1), respectively. On the other hand, carboxypeptidase B did not hydrolyze IQF substrates. The newly developed IQF substrate, Nma-Phe-His-Lys(Dnp), is a valuable tool for ACE and carboxypeptidase studies.

Chitinase from Autographa californica multiple nucleopolyhedrovirus: rapid purification from Sf-9 medium and mode of action.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second ß-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [(GlcNAc)(n), n=4, 5, and 6], producing the ß-anomer of (GlcNAc)2. The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid ß-chitin, producing only (GlcNAc)2. The AcMNPV chitinase processively hydrolyzes solid ß-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.

Purification and characterization of aspartic protease derived from Sf9 insect cells.

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS-PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K(m) of 0.85 µM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) µM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM.

The occurrence of renin inhibitor in rice: isolation, identification, and structure-function relationship.

We found renin inhibitory activity in rice. The physico-chemical data on the isolated inhibitors were identical to those of oleic acid and linoleic acid. Oleic acid and linoleic acid competitively inhibited renin activity, with K(i) values of 15.8 and 19.8 microM respectively. Other unsaturated free fatty acids also inhibited renin activity, but saturated fatty acids had no effect on it.

Direct production of L-ornithine from casein by commercial digestive enzymes and in situ activated arginase.

There is a great demand for L-ornithine, which is used as a dietary supplement, and in the pharmaceutical industry. In the present study, when milk casein was hydrolyzed at 37 degrees C by using commercial digestive enzymes, namely, Pancreatin F and Protease A, a significant accumulation of L-ornithine in the hydrolysate and the simultaneous disappearance of L-arginine was noted. In a radiometric assay, transient but distinct arginase activity, which was sufficiently high for L-ornithine production, was detected in the hydrolysate for a certain period during casein hydrolysis. On the basis of the results of the enzymatic analyses, arginase was thought to be proteolytically generated from an inactive precursor, which may generally be contained in Pancreatin F, and ultimately degraded by further proteolysis. This conversion process using the above-mentioned digestive enzymes is useful for the production of L-ornithine directly from protein sources that are abundant in nature.

Inhibition of human renin activity by saponins.

Renin is the most important enzyme in the renin-angiotensin system. Our previous study led to the identification of soyasaponin I, the first renin inhibitor isolated from soybean. In the present study, the effects of saponins and sapogenols on human renin activities were investigated. Soyasaponins I and II, glycyrrhizin, monoglucuronyl glycyrrhetic acid (MGGA), chikusetsusaponin IV, and Kochia scoparia fruit saponins (momordins) were found to inhibit renin activity. On the other hand, sapogenols (soyasapogenol B and glycyrrhetic acid), saikosaponins b2 and c, and ginsenoside Rb(1) had no effect on renin activity. These results clearly indicate that the 3-O-beta-dglucopyranosiduronic moiety in saponins (glucuronide saponin) is essential for renin inhibition.

Prorenin processing enzyme (PPE) produced by Baculovirus-infected Sf-9 insect cells: PPE is the cysteine protease encoded in the acMNPV gene.

In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610-2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.

Isolation of human renin inhibitor from soybean: soyasaponin I is the novel human renin inhibitor in soybean.

We found human renin inhibitory activity in soybean and isolated the active compound, soybean renin inhibitor (SRI). The physico-chemical data on the isolated SRI were identical with those of soyasaponin I. SRI showed significant inhibition against recombinant human renin, with an IC(50) value of 30 microg/ml. Kinetic studies with SRI indicated partial noncompetitive inhibition, with a K(i) value of 37.5 microM. On the other hand, SRI weakly inhibited pepsin, papain, and bromeline activities, but did not inhibit other proteinases, such as trypsin, kallikrein, angiotensin converting enzyme, and aminopeptidase M. Moreover, a significant (p<0.05) decrease in the systolic blood pressure of spontaneously hypertensive rats was observed when partially purified SRI was orally administrated at 40 mg/kg/d for 7 weeks. This is the first demonstration of a renin inhibitor from soybean, soyasaponin I.

Analysis of inactivation of AcMNPV under various conditions by the ELVA method.

Inactivation of baculoviruses was examined under various conditions by ELVA, which enzymatically labels the virus envelope with radioisotopes and detects the labeled viruses by host cell-specific binding. When baculoviruses were incubated in a 50-ml culture tube, they rapidly lost infective ability by more than 75% of the initial titer in 2 h even at an insect cell cultivation temperature of 27 degrees C. Altering pH from neutral significantly decreased the virus titer. Repetition of freezing and thawing of baculovirus solutions decreased the virus titer, but the addition of DMSO and glycerol prevented inactivation to some extent.

High-level expression of recombinant active human renin in Sf-9 cells: rapid purification and characterization.

Recombinant human (rh) renin was expressed in Sf-9 insect cells. Baculovirus-infected Sf-9 cells produced active rh-renin in the late stage of cultivation. The rh-renin was purified after 5 d of culture by two steps of column chromatography. Approximately 0.61 mg of pure rh-renin was obtained from 200 ml of culture medium with a yield of 35.3%.

A novel method for in vitro radiolabeling and testing enveloped viruses by phosphatidylethanolamine N-methyltransferase and host cell-specific binding.

The present study developed a novel virus labeling and testing method, referred to as an envelope-labeled virus assay (ELVA), in which virus envelope is labeled in vitro by the action of phosphatidylethanolamine N-methyltransferase (PEMT) and tested through a host cell-specific binding. A recombinant strain (vGFPuv) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Spodoptera frugiperda (Sf-9) insect cells were used as a model of viruses and host cells, respectively. The labeling mixture, which contained PEMT, [methyl-3H]S-adenosylmethionine (SAM), and a trace amount of detergent Triton X-100, brought about little change in virus titer of vGFPuv on a 1-h incubation, but was so toxic to Sf-9 cells as to immediately cause cell death. After being incubated with vGFPuv, therefore, the labeling mixture was neutralized by adsorptive removal of PEMT and Triton X-100 before Sf-9 cells were contacted with the mixture to extract the virus. The Sf-9 cells were then washed with a phosphate buffered saline (PBS), and lipid extracts with a 1% SDS solution were subjected to a liquid scintillation analysis for the determination of labeling efficiency. As a result, a significant amount of radioactivity was determined in the extracts, demonstrating the validity of ELVA for labeling and testing enveloped viruses. The conditions for the PEMT reaction and cell-virus binding were examined, and the lower detection limit of AcMNPV by ELVA was found to lie in the order of 10(3) plaque forming unit (pfu) per milliliter. Since the labeling reaction and detection of virus are based on neither immunological nor genetic characteristics of virus, ELVA is also expected to be a convenient and comprehensive test of other enveloped viruses.

Polymer-coated liposomes resistant to enzymatic hydrolysis.

A polymerizable electrolyte, 2-aminoethyl 1,6-heptadien-4-yl phosphate (AEHDP), which has the same hydrophilic head group as naturally occurring phospholipids, was prepared. Five equivalents of AEHDP were added to a suspension of liposomes (closed bilayer vesicles made of phospholipids) and layered on the liposomes. After polymerization by UV irradiation, the resulting polymer-coated liposomes were resistant to hydrolysis of their constituent phospholipids by phospholipase A2.

Adsorption of Cu and Mn on covalently cross-linked alginate gel beads.

The covalently cross-linked alginate gel beads were prepared by the reactions of Ca(2+)-doped alginate gel beads, which were formed by spraying a viscous alginate solution into a calcium chloride solution, with cyanogen bromide and following 1,6-diaminohexane. The cross-linking of alginate matrix decreased the mean bead diameter by about 30% and made the beads durable in some extent under alkaline conditions. The adsorption of metal ions on the covalently cross-linked alginate gel beads was rapid and reached at equilibrium within 30 min at 25 degrees C. Adsorption isotherms of Cu(II), Mn(II), and Ca2+ on the beads possessed a stepwise shape, which was firstly determined by Rorrer et al. [Ind. Eng. Chem. Res. 32 (1993) 2170] for cross-linked chitosan gel beads and explained by a pore-blockage mechanism. Higher selectivity was determined against Cu(II) over Mn(II) and Ca2+, especially at a low concentration region. These metal adsorption profiles for the covalently cross-linked alginate gel beads was almost the same as those for the un-cross-linked beads, indicating that the cross-linking reactions were performed without interfering the adsorption characteristics of alginate gel beads.

Preparation of alginate-chitosan hybrid gel beads and adsorption of divalent metal ions.

Naturally occurring polysaccharides such as alginic acid and chitosan have been recognized as one of the most effective adsorbents to eliminating low levels of heavy metal ions from waste water stream. The present study intended to use alginic acid and chitosan simultaneously, which are expected to form a rigid matrix structure of beads due to electrostatic interaction between carboxyl groups on alginic acid and amino groups on chitosan, and to prepare alginate-chitosan hybrid gel beads. This could be achieved for the first time by using water-soluble chitosan, which was obtained by deacetylating chitin to 36-39% degree. The water-soluble chitosan dissolved in water could remain in solution in the presence of sodium alginate, and the homogeneous solution of chitosan and alginate was dispensed into a CuCl2 solution to give gel bead particles. The resultant beads were then reinforced by a cross-linking reaction of aldehyde groups on glutaraldehyde with amine groups on the chitosan. The cross-linking reaction made the beads durable under acidic conditions. The adsorption of Cu(II), Co(II), and Cd(II) on the beads was significantly rapid and reached at equilibrium within 10 min at 25 degrees C. Adsorption isotherms of the metal ions on the beads exhibited Freundlich and/or Langmuir behavior, contrary to gel beads either of alginate or chitosan showing a step-wise shape of adsorption isotherm.