Platelet-rich plasma does not accelerate the healing of damaged muscle following muscle strain.

Although platelet-rich plasma (PRP) has been widely used regardless of the severity of muscle strain, there have been very few basic studies in which its effects on muscle injury were examined by using models that accurately mimic the clinical muscle strain injury process. Therefore, the aim of this study was to confirm by physiological and structural analyses whether PRP purified by a general preparation method has a muscle healing effect on muscle damage caused by eccentric contraction (ECC). Male Wistar rats were subjected to muscle injury induced by ECC in bilateral plantar flexor muscles using electrical stimulation and an automatically dorsiflexing footplate. The rats were randomly assigned to three groups by type of injection: phosphate-buffered saline (PBS), leukocyte-poor PRP (LP-PRP), or leukocyte-rich PRP (LR-PRP) injection into gastrocnemius muscles three times at weekly intervals. The platelet concentrations of the LP-PRP and LR-PRP were three to five times higher than that of whole blood. The recovery process of torque strength in the plantar flexor muscle, signal changes in MRI images, and histological evaluation 3 weeks after injury showed no obvious differences among the three groups, and every muscle recovered well from the injury without marked fibrosis. The results that neither LP-PRP nor LR-PRP was found to accelerate healing of muscle injuries suggested that conventional preparation and use of PRP for simple muscle injuries caused by muscle strain should be carefully considered, and further basic research using models that accurately mimic clinical practice should be carried out to determine the optimal use of PRP.

Skeletal muscle endurance declines with impaired mitochondrial respiration and inadequate supply of acetyl-CoA during muscle fatigue in 5/6 nephrectomized rats.

Chronic kidney disease (CKD)-related cachexia increases the risks of reduced physical activity and mortality. However, the physiological phenotype of skeletal muscle fatigue and changes in intramuscular metabolites during muscle fatigue in CKD-related cachexia remain unclear. In the present study, we performed detailed muscle physiological evaluation, analysis of mitochondrial function, and comprehensive analysis of metabolic changes before and after muscle fatigue in a 5/6 nephrectomized rat model of CKD. Wistar rats were randomized to a sham-operation (Sham) group that served as a control group or a 5/6 nephrectomy (Nx) group. Eight weeks after the operation, in situ torque and force measurements in plantar flexor muscles in Nx rats using electrical stimulation revealed a significant decrease in muscle endurance during subacute phase related to mitochondrial function. Muscle mass was reduced without changes in the proportions of fiber type-specific myosin heavy chain isoforms in Nx rats. Pyruvate-malate-driven state 3 respiration in isolated mitochondria was impaired in Nx rats. Protein expression levels of mitochondrial respiratory chain complexes III and V were decreased in Nx rats. Metabolome analysis revealed that the increased supply of acetyl CoA in response to fatigue was blunted in Nx rats. These findings suggest that CKD deteriorates skeletal muscle endurance in association with mitochondrial dysfunction and inadequate supply of acetyl-CoA during muscle fatigue.NEW & NOTEWORTHY Mitochondrial dysfunction is associated with decreased skeletal muscle endurance in chronic kidney disease (CKD), but the muscle physiological phenotype and major changes in intramuscular metabolites during muscle fatigue in CKD-related cachexia remain unclear. By using a 5/6 nephrectomized CKD rat model, the present study revealed that CKD is associated with reduced tetanic force in response to repetitive stimuli in a subacute phase, impaired mitochondrial respiration, and inadequate supply of acetyl-CoA during muscle fatigue.

Ultrastructural Assessment and Proteomic Analysis in Myofibrillogenesis in the Heart Primordium After Heartbeat Initiation in Rats.

Myofibrillogenesis is an essential process for cardiogenesis and is closely related to excitation-contraction coupling and the maintenance of heartbeat. It remains unclear whether the formation of myofibrils and sarcomeres is associated with heartbeat initiation in the early embryonic heart development. Here, we investigated the association between the ultrastructure of myofibrils assessed by transmission electron microscopy and their proteomic profiling assessed by data-independent acquisition mass spectrometry (DIA-MS) in the rat heart primordia before and after heartbeat initiation at embryonic day 10.0, when heartbeat begins in rats, and in the primitive heart tube at embryonic day 11.0. Bundles of myofilaments were scattered in a few cells of the heart primordium after heartbeat initiation, whereas there were no typical sarcomeres in the heart primordia both before and after heartbeat initiation. Sarcomeres with Z-lines were identified in cells of the primitive heart tube, though myofilaments were not aligned. DIA-MS proteome analysis revealed that only 43 proteins were significantly upregulated by more than 2.0 fold among a total of 7,762 detected proteins in the heart primordium after heartbeat initiation compared with that before heartbeat initiation. Indeed, of those upregulated proteins, 12 (27.9%) were constituent proteins of myofibrils and 10 (23.3%) were proteins that were accessories and regulators for myofibrillogenesis, suggesting that upregulated proteins that are associated with heartbeat initiation were enriched in myofibrillogenesis. Collectively, our results suggest that the establishment of heartbeat is induced by development of bundles of myofilaments with upregulated proteins associated with myofibrillogensis, whereas sarcomeres are not required for the initial heartbeat.

Enhanced glucose metabolism through activation of HIF-1α covers the energy demand in a rat embryonic heart primordium after heartbeat initiation.

The initiation of heartbeat is an essential step in cardiogenesis in the heart primordium, but it remains unclear how intracellular metabolism responds to increased energy demands after heartbeat initiation. In this study, embryos in Wistar rats at embryonic day 10, at which heartbeat begins in rats, were divided into two groups by the heart primordium before and after heartbeat initiation and their metabolic characteristics were assessed. Metabolome analysis revealed that increased levels of ATP, a main product of glucose catabolism, and reduced glutathione, a by-product of the pentose phosphate pathway, were the major determinants in the heart primordium after heartbeat initiation. Glycolytic capacity and ATP synthesis-linked mitochondrial respiration were significantly increased, but subunits in complexes of mitochondrial oxidative phosphorylation were not upregulated in the heart primordium after heartbeat initiation. Hypoxia-inducible factor (HIF)-1α was activated and a glucose transporter and rate-limiting enzymes of the glycolytic and pentose phosphate pathways, which are HIF-1α-downstream targets, were upregulated in the heart primordium after heartbeat initiation. These results suggest that the HIF-1α-mediated enhancement of glycolysis with activation of the pentose phosphate pathway, potentially leading to antioxidant defense and nucleotide biosynthesis, covers the increased energy demand in the beating and developing heart primordium.

Response to exercise-induced blood pressure elevation is blunted in wrist-cuff automated oscillometric measurement in healthy young college students.

BACKGROUND: A wrist-cuff automated oscillometric device is portable and useful for self-monitoring of blood pressure (BP) at home and outdoors when an upper arm device is not available. Although the height of the forearm in wrist BP measurement is acknowledged to be the major cause of measurement error, it remains unclear whether exercise affects subsequent wrist BP measurement. METHODS AND RESULTS: Ninety-seven healthy college students (median age of 20 years with an age range of 19 to 36 years, 70.1% males) participated in this study. Care was taken to keep the position of the wrist at a level near the upper arm level in BP measurement. At rest, BP measured by a wrist-cuff oscillometric device (Omron HEM-6183) was generally acceptable when it was compared with BP measured by an upper arm oscillometric device (Omron HEM-7130-HP) and with BP measured by the auscultatory method using a mercury sphygmomanometer. However, the ratio of systolic BP measured by oscillometric devices just after a two-step exercise test to that before exercise on the wrist (1.22 ± 0.14) was significantly lower than the ratio on the upper arm (1.27 ± 0.14), and the difference was significantly correlated with exercise-induced increase in pulse rate (Spearman's ρ = 0.23), suggesting a possible role of autonomic nerve activity in the blunted response to exercise-induced BP elevation in wrist BP measurement. CONCLUSIONS: The results indicate that the blunted response to exercise-induced BP elevation should be considered in wrist BP measurement when using a wrist-cuff oscillometric device.

Antinociceptive effects of hyaluronic acid on monoiodoacetate-induced ankle osteoarthritis in rats.

PURPOSE: Ankle osteoarthritis (OA) causes significant pain and debilitation; yet, its underlying mechanisms remain unclear. Clinically, hyaluronic acid (HA) is widely used to treat OA. The present study aimed to investigate the roles of HA in pain-related behavior, joint function, swelling, and pathological changes in cartilage in a rat model of monoiodoacetate (MIA)-induced ankle OA. MATERIALS AND METHODS: Male Sprague Dawley rats were assigned to three experimental groups as follows: 1) MIA rats injected with 1 mg MIA in the right tibiotarsal joint for two consecutive days; 2) sham rats injected with saline instead of MIA; and 3) MIA-HA rats injected with HA in the tibiotarsal joint at 7, 14, and 21 days after MIA injection. Joint swelling, range of motion (ROM), and pain-related behavior were evaluated 1 day before and on the 7th, 14th, 21st, and 28th day after MIA or saline injection. Pathological changes in the ankle joint were assessed 28 days after MIA or saline injection. RESULTS: No significant difference in the degree of ankle swelling or ROM reduction was observed between MIA rats and MIA-HA rats. However, compared with those in MIA rats, mechanical and thermal hypersensitivity was significantly reduced and stride length significantly improved in MIA-HA rats. Histologic analysis revealed that cartilage degeneration was significantly suppressed in MIA-HA rats compared with that in MIA rats, reflecting the chondroprotective effects of HA. CONCLUSION: HA improved pain-related behavior and stride length and suppressed MIA-induced cartilage degeneration. HA may thus inhibit OA progression and suppress peripheral and/or central sensitization.

Analgesic effects of calcitonin on radicular pain in male rats.

PURPOSE: Radicular pain is a frequently observed symptom of lumbar disk herniation or lumbar spinal canal stenosis. Achieving radicular pain relief is difficult. This type of pain may progress to chronic neuropathic pain. Calcitonin (elcatonin [eCT]) has been used mainly for hypercalcemia and pain associated with osteoporosis. The purpose of this study was to investigate analgesic effects of repeated eCT administration on radicular pain in male rats and changes in mRNA-expression levels of voltage-dependent sodium channels in the dorsal root ganglion (DRG). METHODS: Seventy male Sprague-Dawley rats were used. A right L5 hemilaminectomy and an L5-L6 partial facetectomy were performed to expose the right L5 nerve root. Under a microscope, the right L5 spinal nerve root was tightly ligated extradurally with 8-0 nylon suture proximally to the DRG to cause radicular pain in rats. Mechanical hyperalgesia, thermal hyperalgesia, and analgesic effects of eCT were compared among rats with radicular pain that received eCT, those that received the vehicle, and sham rats that received the vehicle. Real-time reverse-transcription PCR was performed to measure mRNA-expression levels of tetrodotoxin-sensitive (NaV1.3 and NaV1.6) and tetrodotoxin-resistant (NaV1.8 and NaV1.9) sodium channels in the DRG. RESULTS: Mechanical and thermal hyperalgesic reactions occurring in rats with radicular pain significantly improved on days 5 and 9 of eCT administration, respectively. In rats with radicular pain, mRNA-expression levels of NaV1.3, NaV1.8, and NaV1.9 increased. After repeated eCT administration, mRNA-expression levels of these sodium channels in rats with radicular pain improved to the same levels as in sham rats. CONCLUSION: The present study demonstrated that repeated systemic eCT administration was effective for radicular pain. No serious side effects of eCT have been reported thus far. Therefore, calcitonin may be a preferred therapeutic option for patients with radicular pain or for those requiring long-term treatment.

The beginning of the calcium transient in rat embryonic heart.

Although many researchers have tried to observe the beginning of the heartbeat, no study has shown the beginning of the calcium transient. Here, we evaluate the beginning of the calcium transient in the Wistar rat heart. We first tried to reveal when the heart of the Wistar rat begins to contract because no previous study has evaluated the beginning of the heartbeat in Wistar rats. Observation of embryos transferred to a small incubator mounted on a microscope revealed that the heart primordium, the so-called cardiac crescent, began to contract at embryonic day 9.99-10.13. Observation of embryos loaded with fluo-3 AM revealed that the beginning of the calcium transient precedes the initiation of contraction which precedes the appearance of the linear heart tube. Nifedipine (1 µM), but not ryanodine (1 µM), abolished the calcium transients. These results indicate that calcium transients in the early embryonic period involve exclusively calcium entry through L-type calcium channels in contrast to the situation in mature hearts. This study provides the first demonstration of the relationship between morphological changes in the heart primordium and the beginning of the calcium transient and contraction.

Enhancement of the nitrogen fixation efficiency of genetically-engineered Rhizobium with high catalase activity.

The vktA catalase gene, which had been cloned from Vibrio rumoiensis S-1T having extraordinarily high catalase activity, was introduced into the root nodule bacterium, Rhizobium leguminosarum bv. phaseoli USDA 2676. The catalase activity of the vktA-transformed R. leguminosarum cells (free-living) was three orders in magnitude higher than that of the parent cells and this transformant could grow in a higher concentration of exogenous hydrogen peroxide (H2O2). The vktA-transformant was inoculated to the host plant (Phaseolus vulgaris L.) and the nodulation efficiency was evaluated. The results showed that the nitrogen-fixing activity of nodules was increased 1.7 to 2.3 times as compared to the parent. The levels of H2O2 in nodules formed by the vktA-transformant were decreased by around 73%, while those of leghemoglobins (Lba and Lbb) were increased by 1.2 (Lba) and 2.1 (Lbb) times compared with the parent. These results indicated that the increase of catalase activity in rhizobia could be useful to improve the nitrogen-fixing efficiency of nodules by the reduction of H2O2 content concomitantly with the enhancement of leghemoglobins contents.

H2O2 tolerance of Vibrio rumoiensis S-1(T) is attributable to the cellular catalase activity.

The extraordinarily high level of H2O2 tolerance of Vibrio rumoiensis strain S-1(T) when compared with the tolerance levels of strain S-4, a probable catalase-deficient derivative of strain S-1(T), was demonstrated by the introduction of 0-100 mM H2O2 during the mid-exponential growth phase. The contribution of catalase to the H2O2 tolerance was also demonstrated by comparing the catalase-deficient mutant Escherichia coli strain UM2 with a UM2 strain, harboring the plasmid pBSsa1, which carried the strain S-1(T) catalase gene vktA. The decomposition rates of 23-25 mM H2O2 that was introduced in the culture fluids of strain S-1(T) and E. coli UM2 harboring pBSsa1 corresponded to the calatase activities of the cells by spectrophotometric measurements. The presence of cell surface catalase was observed by immunoelectron microscopy, using an antibody for intracellular catalase in strain S-1(T). The high level of H2O2 tolerance of strain S-1(T) was attributable to the catalase activity of the cells. Cell surface catalase is considered to contribute to the catalase activity of strain S-1(T) cells.

Cytochrome c-552 from gram-negative alkaliphilic Pseudomonas alcaliphila AL15-21T alters the redox properties at high pH.

A soluble class I cytochrome c of an alkaliphile was purified and characterized, and its primary structure was determined. This is the first example of a soluble class I cytochrome c in alkaliphiles. Cells the alkaliphilic gram-negative bacterium Pseudomonas alcaliphila AL15-21(T) grown at pH 10 had a soluble cytochrome c content that was more than twofold that of strain AL15-21(T) cells grown at pH 7 under air-limited conditions. Cytochrome c-552, a soluble cytochrome c with a low molecular weight, was purified from strain AL15-21(T) cells grown at pH 10 under air-limited conditions. Cytochrome c-552 had a molecular mass of 7.5 kDa and exhibited an almost fully reduced state in the resting form, which exhibited absorption maxima at wavelengths of 552, 523 and 417 nm. In the oxidized state, it exhibited an absorption maximum at 412 nm when it was oxidized by ferricyanide, its isoelectric point (pI) was 4.3 and it contained one heme c as a prosthetic group. Cytochrome c-552 was autoreduced at pH 10, and the autoreduction was reproducible. On the other hand, the autoreduction of cytochrome c-552 was not observed at pH 7.0. When pH was increased from 7.0 to 8.3, its midpoint redox potentials (E(m) values) increased from +228 mV to +276 mV as determined by redox titrations, and from +217 mV to +275 mV as determined by cyclic voltammetric measurements. The amino acid sequence deduced by cytochrome c-552 gene analysis revealed that the sequence consists of 96 residues, including 19 residues as an amino-terminal signal peptide. A phylogenetic tree based on amino acid sequence indicated that the protein belongs to group 4, cytochrome c(5) in class I cytochrome c.

Relationship between the size of the bottleneck 15 A from iron in the main channel and the reactivity of catalase corresponding to the molecular size of substrates.

A catalase that exhibits a high level of activity and a rapid reaction with organic peroxides has been purified from Exiguobacterium oxidotolerans T-2-2T (EKTA catalase). The amino acid sequence of EKTA catalase revealed that it is a novel clade 1 catalase. Amino acid residues in the active site around the protoheme are conserved in the primary structure of EKTA catalase. Although the general interactions of molecules larger than hydrogen peroxide with catalases are strongly inhibited because of the selection role of long and narrow channels in the substrate reaching the active site, the formation rate of reactive intermediates (compound I) in the reaction of EKTA catalase with peracetic acid is 77 times higher than that of bovine liver catalase (BLC) and 1200 times higher than that of Micrococcus luteus catalase (MLC). The crystal structure of EKTA catalase has been determined and refined to 2.4 A resolution. The main channel structure of EKTA catalase is different from those of BLC and MLC. The rate constant of compound I formation in catalases decreased with an increase in the molecular size of the substrate. For EKTA catalase with a larger bottleneck 15 A from the iron (entrance of narrow channel) in the main channel, a lower rate of reduction in compound I formation rate with an increase in the molecular size of substrates was found. The increase in the rate constant of compound I formation in these catalases was directly proportional to the increase in the size of the bottleneck in the main channel when molecules of substrates larger than H2O2, such as organic peroxides, are used in the reaction. The results indicate that the size of the bottleneck in the main channel in catalase is an important factor in defining the rate of compound I formation corresponding to the molecular size of the substrates, and this was demonstrated. The Leu149-Ile180 and Asp109-Met167 combinations at the entrance of the narrow channel in EKTA catalase determine the size of the bottleneck, and each atom-to-atom distance for the combination of residues was larger than those of corresponding combinations of amino acid residues in BLC and MLC. The combination of these four amino acids is quite specific in EKTA catalase as compared with the combinations in other catalases in the gene database (compared with more than 432 catalase genes in the database).

Cultivation of microorganisms in the cultural medium made from squid internal organs and accumulation of polyunsaturated fatty acids in the cells.

The disposal and more efficient utilization of marine wastes is becoming increasingly serious. A culture media for microorganisms has been prepared from squid internal organs that are rich in polyunsaturated fatty acids (PUFAs). Both freshwater and marine bacteria grew well in this medium and some bacteria accumulated PUFAs in their lipids, suggesting uptake of exogenous PUFAs. Higher PUFA accumulations were observed in Escherichia coli mutant cells defective either in unsaturated fatty acid biosynthesis or fatty acid degradation, or both, compared to those without these mutations. Therefore, PUFA accumulation in cells can be improved by genetic modification of fatty acid metabolism in the bacteria. Squid internal organs would be a good source of medium, not only for marine bacteria but also for freshwater bacteria, and that this process may be advantageous to make efficient use of the fishery wastes and to produce PUFA-containing microbial cells and lipids.