RTA-dh404 decreased oxidative stress in mice ischemic limbs and augmented efficacy of therapeutic angiogenesis by intramuscular injection of adipose-derived regenerative cells in the limbs.

Although an intramuscular injection of angiogenic cells to ischemic limbs with peripheral artery disease is a therapeutic option to rescue patients by augmenting neovascularization in the limbs, oxidative stress in the limbs may accelerate apoptosis of the injected cells and thereby reduce the therapeutic effect. In this study involving mice with ischemic lower limbs, whether daily oral administration of RTA-dh404, which is an activator of nuclear factor erythroid 2-related factor 2 (Nrf2) with antioxidant activity, could reduce oxidative stress in the limbs and suppress apoptosis of adipose-derived regenerative cells (ADRCs) injected in the limbs, eventually augmenting neovascularization in the limbs, was evaluated. The tissue expression of Nrf2 and concentrations of total antioxidant capacity and superoxide dismutase in the mice ischemic limbs were higher in the RTA-dh404-treated mice than in the control treated mice, and oxidative stress in the limbs of the RTA-dh404 treated mice was decreased. The day after an intramuscular injection of human ADRCs into ischemic lower limbs of immunodeficient mice, the number of apoptotic ADRCs in the ischemic limbs was decreased by approximately 25% in the RTA-dh404-treated mice compared to the control mice. Fourteen days after cell injection, neovascularization and the salvage ratio were increased by approximately 10% and 63%, respectively, in the ischemic limbs in the RTA-dh404-treated mice compared to the control mice. Pretreatment of ischemic limbs by daily oral administration of RTA-dh404 may augment the effect of therapeutic angiogenesis using an intramuscular injection of ADRCs into the ischemic limbs.

Safety and Efficacy of a Bodyweight Exercise Training Program in Symptomatic Patients With Severe Aortic Valve Stenosis.

Conventional exercise therapy including aerobic and resistance training is desirable for cardiovascular disease, whereas it is generally considered contraindicated for symptomatic severe aortic valve stenosis (AS). This study aimed to evaluate the safety and efficacy of bodyweight resistance exercise training (BRET), which is low-intensity exercise training in symptomatic patients with severe AS. A BRET program consisting of 8 exercises was performed 3 times a week by patients with AS with physical therapists. For the 78 symptomatic patients with severe AS, the median aortic valve area and mean transaortic valve pressure gradient were 0.56 cm2 and 48.9 mm Hg, respectively; none showed any harmful changes in blood pressure or heart rate in 11 sessions of the BRET program. There were no adverse events during hospitalization. Meanwhile, Barthel's Index score significantly improved at the time of hospital discharge. In conclusion, the BRET program in this study did not appear to cause harmful changes in hemodynamics during the program or adverse events during hospitalization, and it improved activities of daily living in symptomatic patients with severe AS, allowing doctors and physical therapists to conduct it safely, with less emotional stress, for cardiac rehabilitation for such patients.

Limited proteolysis by a prostatic endopeptidase, the sperm-activating factor initiatorin, regulates the activation of pro-carboxypeptidase B in the seminal fluid of the silkworm, Bombyx mori.

A prostate trypsin-like serine endopeptidase called initiatorin (BmIni) is an essential factor in triggering the sperm maturation response of the silkworm, Bombyx mori. BmIni has been predicted to specifically cleave the carboxyl side of two consecutive arginine residues present in certain seminal plasma and sperm proteins, but the actual substrates are still unknown. In an attempt to elucidate the molecular mechanism underlying the sperm maturation signaling pathway, in this study, we examined whether BmIni activates the seminal carboxypeptidase B (BmCPB) protein through specific degradation. First, we confirmed in vitro that the inactive BmCPB present in unmated male vesicula (v.) seminalis is activated by treatment with BmIni or trypsin. Molecular cloning of the gene encoding the seminal BmCPB protein has shown that BmCPB is produced as a secreted proenzyme and may be activated after a trypsin-like protease cleaves the boundary between the prodomain and the enzyme site. In support of these findings, both trypsin and BmIni significantly activated recombinant Pro-BmCPB, which was successfully expressed and purified as a proenzyme in Escherichia coli; moreover, two specific cleavage forms appeared in the activation by BmIni that did not appear in that by trypsin. Therefore, a recombinant protein with a mutated diarginine motif (Arg109-Arg110), which is presumed to be a pre-cleavage site of BmCPB based on its high homology with bovine CPB, was prepared and treated with BmIni. As a result, the two specific degraded peptides were no longer observed, and simultaneously the activation was suppressed. Taken together, these findings lead to the conclusion that zymogen BmCPB, which is synthesized and secreted in male reproductive organs, is activated by sequence-dependent proteolysis by BmIni during ejaculation and in the female reproductive organs, providing a clue to the mechanism underlying seminal plasma and/or sperm protein degradation by BmIni in the sperm maturation cascade of B. mori.

Limiting the Pitch Count in Youth Baseball Pitchers Decreases Elbow Pain.

BACKGROUND: Reducing the number of pitches thrown is regarded as the most effective way to prevent throwing injuries in youth baseball pitchers. However, few studies have compared the effectiveness of limiting the pitch count versus the limiting the number of innings pitched in terms of elbow injuries. HYPOTHESIS: We hypothesized that, compared with inning limits, pitch count limits would lead to greater decreases in elbow pain, range of motion deficits, positive moving valgus stress test results, and the risk of capitellar osteochondritis dissecans (OCD). STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: This study retrospectively reviewed baseball pitchers aged 8 to 12 years in 2017 and 2018. Inning and pitch count limits in games were set to a daily maximum of 7 innings in 2017 and 70 pitches in 2018. Elbow pain, range of motion, and moving valgus stress test results were evaluated. The presence of capitellar OCD was assessed on ultrasonographic and radiographic images. RESULTS: A total of 352 pitchers in 2017 and 367 pitchers in 2018 participated. The mean pitch count per game was lower in the pitch count limit (CL) group (52.5 ± 16.0) than in the inning limit (IL) group (98.2 ± 19.5) (P < .001). Compared with the IL group, the CL group had significantly lower rates of elbow pain (40.9% vs 31.9%, respectively; P = .01) and reduced flexion (19.0% vs 10.6%, respectively; P = .001). Multivariate analysis revealed a significant association between elbow pain and age in both the IL and the CL groups (P < .0001 and P = .02, respectively) and between OCD and elbow pain in the CL group (P = .04). CONCLUSION: A pitch count limit of ≤70 pitches per day for baseball pitchers ≤12 years could be more protective against elbow pain and reduced flexion than a limit of ≤7 innings per day, but it may not be effective for reducing the risk of capitellar OCD.

Success in the acquisition of Bombyx mori sperm motility is influenced by the extracellular production of nitric oxide (NO) in the presence of seminal fluid nitric oxide synthase (NOS).

A trypsin-like protease called initiatorin is known to initiate sperm motility in the silkworm, Bombyx mori, but little is known about the signaling events leading to sperm flagellar beating. The aim of this study was to investigate whether this mechanism of sperm motility activation involves the signaling transmitter nitric oxide (NO). NO is produced from the amino acid L-arginine by the enzyme action of nitric oxide synthase (NOS; EC 1.14.13.39). Simple treatment of quiescent sperm with an NO donor (SNAP or NOC7) in vitro did not lead to activation of motility. Nevertheless, initiatorin- or trypsin-induced motility was blocked by pretreatment of sperm with either the NOS inhibitor L-NAME or NO scavenger carboxy-PTIO. These observations suggested that NO may play important physiological roles in the acquisition of sperm motility under the in vitro condition used here. Then, we investigated whether NO synthesis would occur in the spermatophore, a capsule containing spermatozoa that is created by the contents of various male reproductive glands and is the site of sperm maturation. The amounts of NO2- and NO3-, stable metabolites of NO, reached maximum values after enclosure in the spermatophore, a time when apyrene spermatozoa acquire vigorous motility. Moreover, RT-PCR and Western blotting analyses of NOS indicated that it is abundantly expressed in glandula (g.) lacteola of the virgin male ejaculatory duct, from which it is secreted to the seminal fluid and transferred to the female during mating. Previous studies demonstrated that free L-arginine is supplied de novo by a specific proteolytic reaction in which initiatorin participates during spermatophore formation (Osanai et al., 1987c). Based on these results, it can be presumed that the mixing of seminal fluid contents from each male reproductive organ during ejaculation induced NO production outside of the spermatid, and exogenous NO stimulated a signaling pathway involved in the activation of silkworm apyrene sperm.

Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori.

Male Bombyx mori has a trypsin-type protease, called initiatorin, in the secretion from the posterior segment of the ejaculatory duct that is thought to be involved in the acquisition of sperm motility, although this inference remains to be demonstrated. Here, we revised the experimental procedures including that for purification and definitely identified the purified initiatorin protein as an activation factor of B. mori sperm by an in vitro study in which we treated isolated spermatozoa with this enzyme. Analysis of cDNA revealed that initiatorin consists of 281 amino acids with sequence similarity to bovine trypsin, and is highly homologous to the ejaculated accessory gland proteins not only of other Lepidoptera but also of Orthoptera. Recombinant initiatorin, expressed in Escherichia coli and purified, also showed proteolytic and sperm-activating activities. RT-PCR and Western blot analyses indicated that initiatorin is abundantly expressed in the glandula (g.) prostatica. It was also shown that pro-initiatorin is synthesized and stored in g. prostatica, and then converted to the mature form upon ejaculation. Fluorogenic peptides with a dibasic sequence were efficiently cleaved by initiatorin, and one such substrate, BOC-Gly-Arg-Arg-MCA, inhibited sperm activation by the extract of g. prostatica. These results delineate the idea that initiatorin has the most suitable protease property as an initiator of the protein degradation cascade in that it releases free arginines, which in turn become an energy resource for sperm motility.

Comparison of efficiency between FLPe and Cre for recombinase-mediated cassette exchange in vitro and in adenovirus vector production.

Cre and FLP recombinases mediate not only specific deletions and insertions, but also the recombinase-mediated cassette exchange (RMCE) reaction, which is used in cell biotechnology including ES cells and mouse genetics. However, comparison of efficiencies for Cre and FLP in RMCE has not been made. We here examined the detailed process of RMCE with Cre and FLP in vitro using mutant loxP 2272 and three mutant FRTs (FRT G, FRT H, and FRT F3) and then quantitatively compared the RMCE reactions in vitro. Interestingly, in the in vitro reactions, the RMCE efficiency of Cre reached a plateau level of approximately 5% and did not proceed further, whereas that of FLPe reached approximately 12-13%, showing that FLPe reached a higher level of efficiency than Cre possibly when they were supplied at a very high concentration. Moreover, we quantitatively compared the production efficiency of E1-deleted adenovirus vector using the RMCE method with Cre or FLP. The results showed that FLPe was again found more efficient than Cre in RMCE reaction. Thus, although Cre is considered more active than, or similar to, FLPe, it may not be necessarily true for RMCE reaction. Possible reasons explaining these results are discussed.

Identification of two arginases generated by alternative splicing in the silkworm, Bombyx mori.

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of arginine to ornithine and urea. Here, we have cloned two arginase cDNAs from the silkworm, Bombyx mori. The analysis of exon/intron structures showed that the two mRNAs named bmarg-r and bmarg-f were generated from a single gene by alternative usage of exons. The bmarg-r and bmarg-f were predicted to encode almost the same amino acid sequences, except that the latter had additional ten N-terminal residues. Recombinant bmARG-r and bmARG-f in Escherichia coli cell lysates were roughly similar to each other in enzymatic characteristics, which did not show large difference from those of arginases assayed by using tissue extracts. Differential RT-PCR experiments and tissue distribution analyses of arginase activity indicated that the bmarg-r gene is expressed in the male reproductive organs, especially in the glandula lacteola and vesicular seminalis, from which it is secreted to the seminal fluid and transferred to the female during copulation, whereas the bmarg-f gene is expressed in the larval and adult nonreproductive organs including the fat body and muscle, where the produced arginase proteins are considered to stay in the cells. Thus, the two silkworm arginase isoforms may have a difference in whether or not the product is excreted out of the cells in which it is synthesized.

Activities of various FLP recombinases expressed by adenovirus vectors in mammalian cells.

FLP, like Cre, is a frequently employed site-specific recombinase. Because wild-type FLP (wtFLP) is thermolabile, a thermostable FLP mutant (FLPe) has been developed for efficient recombination of FLP in studies using mammalian cells and animals. FLPe and wtFLP have been compared in multiple assays in vitro and in vivo, and in mouse genetics, FLPe has been shown to be very effective like Cre. Here we show an adenovirus vector (AdV) system to be valuable for quantitative measurements of the enzyme activity in mammalian cells and, using this system, precisely compare activities of wtFLP and FLPe. Unexpectedly, we found that the recombination efficiency of FLPe enzyme was lower on a molar basis than that of wtFLP even at 37 degrees C and, consequently, that the higher recombination yield per transduced AdV genome expressing FLPe compared to wtFLP was due not to inherently higher enzyme activity, but rather to higher steady-state levels of FLPe by its thermostability. Therefore, trying to increase FLPe levels further, we generated a "humanized" FLPe (hFLPe) gene with codon usage optimized for mammals. hFLPe produced about 10-fold more FLPe enzyme in transfection experiments than FLPe, as expected. However, hFLPe-expressing AdV was unstable and could not be prepared without deletion, suggesting that a subtle deleterious effect of FLP on 293 cells may exist. With hFLPe-expressing AdV thus unavailable, of the AdV constructs tested, AdV-expressing FLPe yielded the most recombined targets, despite the lower recombination efficiency of FLPe per enzyme molecule compared with that of wtFLP. We found hFLPe to be valuable for plasmid transfection, and its properties are probably suitable for experiments involving cell lines and transgenic mice.