Search

Amyloid fibrils of β2-microglobulin (β2m) can cause dialysis-related amyloidosis. Here, the authors show that a decrease in serum albumin levels in long-term dialysis deteriorates the inhibitory effects of serum milieux on supersaturation-limited amyloid formation of β2m, suggesting that macromolecular crowding protects the onset of amyloidosis.

Much effort has been devoted to elucidate mechanisms of amyloid fibril formation using various kinds of additives, such as salts, metals, detergents, and biopolymers. Here, we review the effects of additives with a focus on polyphosphate (polyP) on amyloid fibril formation of β2-microglobulin (β2m) and α-synuclein (αSyn). PolyP, consisting of up to 1,000 phosphoanhydride bond-linked phosphate monomers, is one of the most ancient, enigmatic, and negatively charged molecules in biology. Amyloid fibril formation of both β2m and αSyn could be accelerated by counter anion-binding and preferential hydration at relatively lower and higher concentrations of polyP, respectively, depending on the chain length of polyP. These bimodal concentration-dependent effects were also observed in salt- and heparin-induced amyloid fibril formation, indicating the generality of bimodal effects. We also address the effects of detergents, alcohols, and isoelectric point precipitation on amyloid fibril formation, in comparison with the effects of salts. Because polyP is present all around us, from cellular components to food additives, clarifying its effects and consequent biological roles will be important to further advance our understanding of amyloid fibrils. This review article is an extended version of the Japanese article, Linking Protein Folding to Amyloid Formation, published in SEIBUTSU BUTSURI Vol. 61, p. 358–365 (2021).

Abstract Parkinson’s disease is a neurodegenerative disease characterized by the formation of neuronal inclusions of α-synuclein in patient brains. As the disease progresses, toxic α-synuclein aggregates transmit throughout the nervous system. No effective disease-modifying therapy has been established, and preventing α-synuclein aggregation is thought to be one of the most promising approaches to ameliorate the disease. In this study, we performed a two-step screening using the thioflavin T assay and a cell-based assay to identify α-synuclein aggregation inhibitors. The first screening, thioflavin T assay, allowed the identification of 30 molecules, among a total of 1262 FDA-approved small compounds, which showed inhibitory effects on α-synuclein fibrilization. In the second screening, a cell-based aggregation assay, seven out of these 30 candidates were found to prevent α-synuclein aggregation without causing substantial toxicity. Of the seven final candidates, tannic acid was the most promising compound. The robustness of our screening method was validated by a primary neuronal cell model and a Caenorhabditis elegans model, which demonstrated the effect of tannic acid against α-synuclein aggregation. In conclusion, our two-step screening system is a powerful method for the identification of α-synuclein aggregation inhibitors, and tannic acid is a promising candidate as a disease-modifying drug for Parkinson’s disease.

BackgroundPhysically active status is an important contributor to individual health. Walking is regarded as commonly accepted exercise for exercise promotion. Particularly, interval fast walking (FW), consisting of alternating between fast and slow walking speeds, has gained popularity from practical viewpoints. Although previous studies have determined the short- and long-term effects of FW programs on endurance capacity and cardiovascular variables, factors affecting these outcomes have not been clarified. In addition to physiological variables, understanding of mechanical variables and muscle activity during FW would be a help to understand characteristics of FW. In the present study, we compared the ground reaction force (GRF) and lower limb muscle activity between fast walking (FW) and running at equivalent speeds.MethodEight healthy men performed slow walking (45% of the maximum walking speed; SW, 3.9 ± 0.2 km/h), FW (85% of the maximum walking speed, 7.4 ± 0.4 km/h), and running at equivalent speeds (Run) for 4 min each. GRF and average muscle activity (aEMG) were evaluated during the contact, braking, and propulsive phases. Muscle activities were determined for seven lower limb muscles: gluteus maximus (GM), biceps femoris (BF), rectus femoris (RF), vastus lateralis (VL), gastrocnemius medialis (MG), soleus (SOL), and tibialis anterior (TA).ResultsThe anteroposterior GRF was greater in FW than in Run during the propulsive phase (p

Noji et al. test link between protein folding and misfolding upon heating and agitation. They show that folding and amyloid formation are separated by the supersaturation barrier of a protein, breakdown of which shifts the protein to the amyloid pathway. This study is useful to the field of protein folding versus self-assembly and amyloidogenesis.

Abstract Blood flow restriction (BFR) during low‐intensity exercise has been known to be a potent procedure to alter metabolic and oxygen environments in working muscles. Moreover, the use of BFR during inter‐set rest periods of repeated sprint exercise has been recently suggested to be a potent procedure for improving training adaptations. The present study was designed to determine the effect of repeated sprint exercise with post‐exercise BFR (BFR during rest periods between sprints) on muscle oxygenation in working muscles. Eleven healthy males performed two different conditions on different days: either repeated sprint exercise with BFR during rest periods between sets (BFR condition) or without BFR (CON condition). A repeated sprint exercise consisted of three sets of 3 × 6‐s maximal sprints (pedaling) with 24s rest periods between sprints and 5 min rest periods between sets. In BFR condition, two min of BFR (100–120 mmHg) for both legs was conducted between sets. During the exercise, power output and arterial oxygen saturation (SpO2) were evaluated. Muscle oxygenation for the vastus lateralis muscle, exercise‐induced changes in muscle blood flow, and muscle oxygen consumption were measured. During BFR between sets, BFR condition presented significantly higher deoxygenated hemoglobin + myoglobin (p

Proteins in either a native or denatured conformation often aggregate at an isoelectric point (pI), a phenomenon known as pI precipitation. However, only a few studies have addressed the role of pI precipitation in amyloid formation, the crystal-like aggregation of denatured proteins. We found that α-synuclein, an intrinsically disordered protein of 140 amino acid residues associated with Parkinson's disease, formed amyloid fibrils at pI (= 4.7) under the low-sodium phosphate conditions. Although α-synuclein also formed amyloid fibrils at a wide pH range under high concentrations of sodium phosphate, the pI-amyloid formation was characterized by marked amyloid-specific thioflavin T fluorescence and clear fibrillar morphology, indicating highly ordered structures. Analysis by heteronuclear NMR in combination with principal component analysis suggested that amyloid formation under low and high phosphate conditions occurred by distinct mechanisms. The former was likely to be caused by the intermolecular attractive charge-charge interactions, where α-synuclein has +17 and −17 charges even with the zero net charge. On the other hand, the latter was caused by the phosphate-dependent salting-out effects. pI-amyloid formation may play a role in the membrane-dependent amyloid formation of α-synuclein, where the negatively charged membrane surface reduces the local pH to pI and the membrane hydrophobic environment enhances electrostatic interactions. The results extend the supersaturation-limited mechanism of amyloid formation: Amyloid fibrils are formed under a variety of conditions of decreased solubility of denatured proteins triggered by the breakdown of supersaturation.

PurposeThis study aimed to examine the effect of applying BFR during rest periods of repeated cycling sprints on muscle oxygenation.MethodsSeven active males performed 5 × 10-s maximal pedaling efforts with 40-s passive rest, with or without BFR application during rest period. BFR was applied for 30 s between sprints (between 5 and 35 s into rest) through a pneumatic pressure cuff inflated at 140 mmHg. Vastus lateralis muscle oxygenation was monitored using near-infrared spectroscopy. In addition, blood lactate concentration and heart rate were also evaluated.ResultsThe BFR trial showed significantly lower oxyhemoglobin (oxy-Hb) and tissue saturation (StO2) levels than the CON trial (P < 0.05). However, power output and blood lactate concentration did not significantly differ between the two trials (P > 0.05).ConclusionApplying BFR during rest periods of repeated cycling sprints decreased muscle oxygenation of active musculature, without interfering with power output during sprints.

Ultrasonication to supersaturated protein solutions forcibly forms amyloid fibrils, thereby allowing the early-stage diagnosis for amyloidoses. Previously, we constructed a high-throughput sonoreactor to investigate features of the amyloid-fibril nucleation. Although the instrument substantiated the ultrasonication efficacy, several challenges remain; the key is the precise control of the acoustic field in the reactor, which directly affects the fibril-formation reaction. In the present study, we develop the optimized sonoreactor for the amyloid-fibril assay, which improves the reproducibility and controllability of the fibril formation. Using β2-microglobulin, we experimentally demonstrate that achieving identical acoustic conditions by controlling oscillation amplitude and frequency of each transducer results in identical fibril-formation behavior across 36 solutions. Moreover, we succeed in detecting the 100-fM seeds using the developed sonoreactor at an accelerated rate. Finally, we reveal that the acceleration of the fibril-formation reaction with the seeds is achieved by enhancing the primary nucleation and the fibril fragmentation through the analysis of the fibril-formation kinetics. These results demonstrate the efficacy of the developed sonoreactor for the diagnosis of amyloidoses owing to the accelerative seed detection and the possibility for further early-stage diagnosis even without seeds through the accelerated primary nucleation.

Purpose: The purpose of this study was to determine the effect of 3 consecutive days of endurance training under hypoxia on muscle damage, inflammation, and performance responses.Methods: Nine active healthy males completed two trials in different periods, consisting of either 3 consecutive days of endurance training under hypoxia [fraction of inspired oxygen (Fio2): 14.5%, HYP] or normoxia (Fio2: 20.9%, NOR). They performed daily 90-min sessions of endurance training consisting of high-intensity endurance interval pedaling [10 × 4-min pedaling at 80% of maximal oxygen uptake (V˙o2max) with 2 min of active rest at 30% of V˙o2max] followed by 30-min continuous pedaling at 60% of V˙o2max during 3 consecutive days (days 1–3). Venous blood sample, muscular performance of lower limb, and score of subjective feelings were determined every morning (days 1–4) to evaluate muscle damage and inflammation. On day 4, subjects performed an incremental exercise test (IET) to evaluate the performance response.Results: Pedaling workload during daily endurance training was significantly lower in the HYP trial (interval exercise: 166 ± 4 W) than in the NOR trial (194 ± 8 W; P < 0.0001). Serum creatine kinase (CK) and high-sensitivity C-reactive protein (hsCRP) concentrations did not significantly change during days 1–4 in either trial. Maximal voluntary contraction (MVC) of knee extension (P < 0.0001) and drop jump (DJ) index (P = 0.004) were significantly decreased with training in both trials, with no significant difference between trials. The muscle soreness and fatigue scores significantly increased in both trials (P < 0.0001). However, the HYP trial showed a significantly lower score of fatigue on day 4 compared with the NOR trial (P = 0.004). Maximal aerobic power output during IET on day 4 did not significantly differ between trials.Conclusion: Three consecutive days of endurance training under hypoxia induced comparable levels of muscle damage, inflammation, and performance responses compared with the same training under normoxia.

The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute’s concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases to its thermodynamic solubility. Soon after the discovery of the prion phenomena, it was recognized that prion disease transmission and propagation share some similarities with the process of crystallization. Subsequent studies exploring the structural and functional association between amyloid fibrils and amyloidoses solidified this paradigm. However, recent studies have not necessarily focused on supersaturation, possibly because of marked advancements in structural studies clarifying the atomic structures of amyloid fibrils. On the other hand, there is increasing evidence that supersaturation plays a critical role in the formation of amyloid fibrils and the onset of amyloidosis. Here, we review the recent evidence that supersaturation plays a role in linking unfolding/folding and amyloid fibril formation. We also introduce the HANABI (HANdai Amyloid Burst Inducer) system, which enables high-throughput analysis of amyloid fibril formation by the ultrasonication-triggered breakdown of supersaturation. In addition to structural studies, studies based on solubility and supersaturation are essential both to developing a comprehensive understanding of amyloid fibrils and their roles in amyloidosis, and to developing therapeutic strategies.

Abstract We investigated performance, energy metabolism, acid–base balance, and endocrine responses to repeated‐sprint exercise in hot and/or hypoxic environment. In a single‐blind, cross‐over study, 10 male highly trained athletes completed a repeated cycle sprint exercise (3 sets of 3 × 10‐s maximal sprints with 40‐s passive recovery) under four conditions (control [CON; 20℃, 50% rH, FiO2: 20.9%; sea level], hypoxia [HYP; 20℃, 50% rH, FiO2: 14.5%; a simulated altitude of 3,000 m], hot [HOT; 35℃, 50% rH, FiO2: 20.9%; sea level], and hot + hypoxia [HH; 35℃, 50% rH, FiO2: 14.5%; a simulated altitude of 3,000 m]). Changes in power output, muscle and skin temperatures, and respiratory oxygen uptake were measured. Peak (CON: 912 ± 26 W, 95% confidence interval [CI]: 862–962 W, HYP: 915 ± 28 W [CI: 860–970 W], HOT: 937 ± 26 W [CI: 887–987 W], HH: 937 ± 26 W [CI: 886–987 W]) and mean (CON: 808 ± 22 W [CI: 765–851 W], HYP: 810 ± 23 W [CI: 765–855 W], HOT: 825 ± 22 W [CI: 781–868 W], HH: 824 ± 25 W [CI: 776–873 W]) power outputs were significantly greater when exercising in heat conditions (HOT and HH) during the first sprint (p

β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the deleterious synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules (e.g., lipids) and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular components, including extracellular matrix proteins.

We compared upper limb muscle oxygenation responses during repeated double-poling sprint exercise in normobaric hypoxia and normoxia. Eight male kayakers completed a repeated double-poling sprint exercise (3 × 3 × 20-s maximal sprints, 40-s passive recovery, 5-min rest) in either hypoxia (HYP, FiO2 = 14.5%) or normoxia (NOR, FiO2 = 20.9%). Power output, muscle oxygenation of triceps brachii muscle (using near infrared spectroscopy), arterial oxygen saturation, and cardiorespiratory variables were monitored. Mean power output tended to be lower (-5.2%; P = 0.06) in HYP compared with NOR, while arterial oxygen saturation (82.9 ± 0.9% vs. 90.5 ± 0.8%) and systemic oxygen uptake (1936 ± 140 vs. 2408 ± 83 mL⋅min-1) values were lower (P < 0.05). Exercise-induced increases in deoxygenated hemoglobin (241.7 ± 46.9% vs. 175.8 ± 27.2%) and total hemoglobin (138.0 ± 18.1% vs. 112.1 ± 6.7%) were greater in HYP in reference to NOR (P < 0.05). Despite moderate hypoxia exacerbating exercise-induced elevation in blood perfusion of active upper limb musculature, power output during repeated double-poling exercise only tended to be lower.

Protein-bound uremic toxins (PBUTs) are difficult to remove using conventional dialysis treatment owing to their high protein-binding affinity. As pH changes the conformation of proteins, it may be associated with the binding of uremic toxins. Albumin conformation at pH 2 to 13 was analyzed using circular dichroism. The protein binding behavior between indoxyl sulfate (IS) and albumin was examined using isothermal titration calorimetry. Albumin with IS, and serum with IS, p-cresyl sulfate, indole acetic acid or phenyl sulfate, as well as serum from hemodialysis patients, were adjusted pH of 3 to 11, and the concentration of the free PBUTs was measured using mass spectrometry. Albumin was unfolded at pH < 4 or >12, and weakened interaction with IS occurred at pH < 5 or >10. The concentration of free IS in the albumin solution was increased at pH 4.0 and pH 11.0. Addition of human serum to each toxin resulted in increased free forms at acidic and alkaline pH. The pH values of serums from patients undergoing hemodialysis adjusted to 3.4 and 11.3 resulted in increased concentrations of the free forms of PBUTs. In conclusion, acidic and alkaline pH conditions changed the albumin conformation and weakened the protein binding property of PBUTs in vitro.

The present study investigated the effects of a combined hot and hypoxic environment on muscle oxygenation during repeated 15-s maximal cycling sprints. In a single-blind, cross-over study, nine trained sprinters performed three 15-s maximal cycling sprints interspersed with 7-min passive recovery in normoxic (NOR; 23°C, 50%, FiO2 20.9%), normobaric hypoxic (HYP; 23°C FiO2 14.5%), and hot normobaric hypoxic (HH; 35°C, FiO2 14.5%) environments. Relative humidity was set to 50% in all trials. The vastus lateralis muscle oxygenation was evaluated during exercise using near-infrared spectroscopy. The oxygen uptake (VO2) and arterial oxygen saturation (SpO2) were also monitored. There was no significant difference in peak or mean power output among the three conditions. The reduction in tissue saturation index was significantly greater in the HH (-17.0 ± 2.7%) than in the HYP (-10.4 ± 2.8%) condition during the second sprint (p < 0.05). The average VO2 and SpO2 were significantly lower in the HYP (VO2 = 980 ± 52 mL/min, SpO2 = 82.9 ± 0.8%) and HH (VO2 = 965 ± 42 mL/min, SpO2 = 83.2 ± 1.2%) than in the NOR (VO2 = 1149 ± 40 mL/min, SpO2 = 90.6 ± 1.4%; p < 0.05) condition. In conclusion, muscle oxygen saturation was reduced to a greater extent in the HH than in the HYP condition during the second bout of three 15-s maximal cycling sprints, despite the equivalent hypoxic stress between HH and HYP. [ABSTRACT FROM AUTHOR]

Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H2SO4, of β2-microglobulin (β2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na2SO4. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of β2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism. [ABSTRACT FROM AUTHOR]

Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonicationdependent a-synuclein (a-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longerchain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate. The second optimum decreased markedly as the polyP length increased. This suggested that although the optima at lower polyP concentrations were caused by interactions between negatively charged phosphate groups and the positive charges of a-syn, the optima at higher polyP concentrations were caused by the Hofmeister salting-out effects of phosphate groups, where the effects do not depend on the net charge. NMR titration experiments of a-syn with tetraphosphate combined with principal component analysis revealed that, at low tetraphosphate concentrations, negatively charged tetraphosphates interacted with positively charged "KTK" segments in four KTKEGV repeats located at the N-terminal region. At high concentrations, hydrated tetraphosphates affected the surface-exposed hydrophilic groups of compact a-syn. Taken together, our results suggest that long-chain polyPs consisting of 60 to 70 phosphates induce amyloid formation at sub-µM concentrations, which are comparable with the concentrations of polyPs in the blood or tissues. Thus, these findings may identify a role for polyPs in the pathogenesis of amyloid-related diseases. [ABSTRACT FROM AUTHOR]

Structural changes of globular proteins and their resultant amyloid aggregation have been associated with various human diseases, such as lysozyme amyloidosis and light-chain amyloidosis. Because many globular proteins can convert into amyloid fibrils in vitro, the mechanisms of amyloid fibril formation have been studied in various experimental systems, but several questions remain unresolved. Here, using several approaches, such as turbidimetry, fluorescence and CD spectroscopy, EM, and isothermal titration calorimetry, we examined the binding of polyphosphates to hen egg-white lysozyme under acidic conditions and observed polyphosphate-induced structural changes of the protein promoting its aggregation. Our data indicate that negatively charged polyphosphates bind to protein molecules with a net positive charge. The polyphosphate-bound, structurally destabilized protein molecules then start assembling into insoluble amorphous aggregates once they pass the solubility limit. We further show that the polyphosphates decrease the solubility limit of the protein and near this limit, the protein molecules are in a labile state and highly prone to converting into amyloid fibrils. Our results explain how polyphosphates affect amorphous aggregation of proteins, how amyloid formation is induced in the presence of polyphosphates, and how polyphosphate chain length is an important factor in amyloid formation. [ABSTRACT FROM AUTHOR]

Repeated sprint exercise in hypoxic condition is effective for improving repeated sprint ability. Moreover, repeated sprint exercise with voluntary hypoventilation at low lung volume (VHL) may be alternative procedures for establishing hypoxic environment in the body. Although the training with VHL caused further increase in repeated sprint ability compared with the same training with normal breath (Woorons et al., 2019), no direct comparison of the physiological responses to repeated sprint exercise between hypoxic condition and VHL was performed. PURPOSE: To compare the physiological responses during repeated sprint exercise using VHL with hypoxic condition. METHODS: Ten male subjects (21.8 ± 0.4 yrs, 168.6 ± 1.5 cm, 65.0 ± 2.2 kg) completed a single session of repeated sprint exercise (three sets of 6x8 s high intensity pedaling at 170% of VO2max) under 3 different conditions, (1) normoxia with normal breathing (NOR; 23°, FiO2 = 20.9%), (2) hypoxia with normal breathing (HYP; 23°, FiO2 = 14.5%), and (3) normoxia with VHL (VHL; 23°, FiO2 = 20.9%) with a single blind, randomized crossover design. Power output during each sprint, arterial oxygen saturation, heart rate, and muscle oxygenation were monitored during the exercise. We also measured muscle blood flow (mBF) and muscle oxygen consumption in vastus lateralis (muscle VO2) immediately after the exercise using venous/arterial occlusion and near infrared spectroscopy. RESULTS: Arterial oxygen saturation was significantly lower in VHL than in NOR. However, arterial oxygen saturation in VHL was significantly higher compared with HYP (NOR: 94.9±0.4%, HYP: 82.8±1.2%, VHL: 90.4±0.5%, p < 0.05). Moreover, muscle oxygen saturation in HYP was significantly lower compared with both VHL and NOR (NOR: 80.1±25.3%, HYP: 66.4±21.0%, VHL: 75.2±23.8%, p < 0.05), but there was no significant difference between VHL and NOR (p > 0.05). Also, mBF and muscle VO2 immediately after exercise did not differ significantly among conditions (p > 0.05). CONCLUSIONS: Although arterial oxygen saturation decreased during repeated sprint exercise with VHL, the exercise-induced decrement was smaller than hypoxic condition. Moreover, adding VHL during repeated sprint exercise did not elicit mBF and muscle VO2. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of the present study was to examine changes in electrolyte and acid-base balance before and after a 60-min of running session between early follicular and luteal phases in female athletes. Methods: Eleven female athletes {age 21.2 ± 0.3 yrs, peak oxygen uptake (VO2peak) 46.3 ± 1.0 ml/kg/min} conducted two different trials which consisted of 60-min of running at 75% of VO2peak during early follicular phase (FOL; days 1-5 of the menstrual cycle) and luteal phase (LUT: days 20-25 of the menstrual cycle). Blood samplings were collected before. immediately. 1 h and 3h after the exercise session. Results: FOL trial presented lower serum estradiol and progesterone concentration than LUT trials 01= 0.002 and p = 0.006. respectively). Blood pH increased immediately after exercise. while blood PCO2 and HCO3 decreased immediately after exercise in both trials. Changes in blood pH, pCO2 and HCO3 did not significantly differ between FOL and LUT trials. Na+ concentrations did not significantly change before and after exercise. K+ concentrations significantly elevated immediately after exercise in both trials. Discussion: These results suggest that moderate intensity running does not affect exercise-induced changes in electrocyte and acid-base balance during different phages of the menstrual cycle. [ABSTRACT FROM AUTHOR]

The effects of aspartate for exercise performance have been investigated since 1950s. Aspartate may improve endurance performance in humans, while no effect on muscular endurance and strength has been observed. Potential mechanisms for improved endurance performance (e.g., attenuated exercise-induced hyperammonemia, promoted muscle glycogen sparing, free fatty acid oxidation and gluconeogenesis) were suggested. However, effect of aspartate on repeated sprint performance is unknown. PURPOSE: The present study was designed to determine the effects of sodium L-aspartate supplementation on repeated cycling sprint performance. METHODS: Fourteen healthy males participated in the present study (22.3 ± 0.3 years, 172.1 ± 1.7 cm, 65.4 ± 2.2 kg). They completed repeated cycling sprint exercise (three sets of 10 x 6 s maximal pedaling with 40 s rest between sprints) in two different trials, consisting of sodium L-aspartate supplementation trial [ASP; consuming 2 x 4 g (8 g in total) of sodium L-aspartate] and placebo trial [PLA; 2 x 4 g (8 g in total) of maltitol] with a double blind, randomized crossover design. Both supplements were granulated and consumed with 200 mL of water twice on each testing day (25 min before starting the first set of exercise and immediately after the first set of exercise). Revolution per minute (rpm) and power output were measured during each sprint. Venous blood samples were collected to determine exercise-induced changes in blood variables (e.g., blood pH, glucose, lactate, bicarbonate and amino acid concentrations). RESULTS: The ASP showed significantly higher blood aspartate, glutamate, alanine, phenylalanine, citrulline, and total amino acid concentrations compared with those in PLA (p < 0.05). Peak rpm for the second set of the exercise was significantly higher in ASP (153 ± 3 rpm) compared with PLA (152 ± 3 rpm, p < 0.05). In addition, peak power output for the second set tended to be higher in ASP (741 ± 32 rpm) than in PLA (734 ± 31 rpm, p = 0.06). Blood pH and bicarbonate concentration tended to be higher in ASP immediately before the third set (pH, p = 0.098) and 60 min after entire exercise (bicarbonate, p = 0.052) compared with PLA. CONCLUSIONS: These results suggest that sodium L-aspartate supplementation could improve power output during repeated sprint exercise. [ABSTRACT FROM AUTHOR]

Purpose: To compare gastric emptying rate after repeated sprint exercise in normoxia, hypoxia, and heat hypoxia. Methods: Eleven male athletes conducted 4 different trials on separate days consisting of 3 exercise trials in normoxia (NOR; 23°C, Fi02 = 20.9%), hypoxia (HYP; 23°C, Fi02 = 14.5%), and heat hypoxia (HH; 35°C, Fi02 = 14.5%), and a rest trial (CON; 23°C, Fi02 = 20.9%). Power output, rectal and skin temperature, and muscle blood flow were measured during or immediately after exercise. We also evaluated gastric emptying rate for 90 min after exercise using 13C-sodium acetate breath test. Results: HH showed significantly higher rectal and skin temperature compared with NOR and HYP. Power output and average muscle blood flow were not different among 3 exercise trials. Gastric emptying was significantly delayed in NOR, HYP, and HH compared with CON, although no difference was observed among 3 exercise trials. Discussion: Exercise-induced increase in muscle blood flow may be related to delayed gastric emptying in exercise trials due to decreased gastric blood flow. Conclusion: Repeated sprint exercise significantly delayed subsequent gastric emptying. However, different environments (hypoxia and heat conditions) during repeated sprint exercise did not affect gastric emptying rate. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to examine the muscle oxygenation to sprint exercise under heat and hypoxic condition. Methods: Nine male track and field sprinters participated. They conducted 3 X 15 s maximal sprint exercise under either "heat and hypoxic" (fraction of inspiratory oxygen (FiO2) : 14.5%, 35°C), "hypoxic" (Fi02:14.5%, 23°C) or "normoxic" (FiO2 :20.9%, 23°C) conditions. We evaluated muscle oxygenation (vastus laterals) during exercise, changes in blood variables, percutaneous oxygen saturation (SpO2), muscle and skin temperature, energy expenditure. Results and Discussion: No significant difference was observed in peak and mean power among the three conditions. Muscle temperature was significantly increased under heat and hypoxic condition than normoxic and hypoxic conditions (P< 0.05). SpO2 was significantly decreased both under "heat and hypoxic" and "hypoxic" conditions (P < 0.05). Blood lactate was significantly increased during exercise (P < 0.05), but no significant difference was observed among three conditions. Tissue oxygen saturation (StO2) was significantly lower in heat and hypoxic condition compared with hypoxic condition (P<0.05). This result suggested that local hypoxia was accelerated during sprint exercise under heat and hypoxic condition. [ABSTRACT FROM AUTHOR]

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes. However, the role of polyP in immune cell dysfunction is not well-understood. In this study, using several biochemical and cell biology approaches, including cytokine assays, immunofluorescence microscopy, receptor-binding assays with quartz crystal microbalance, and dynamic light scanning, we investigated the effect of polyP on in vitro lipopolysaccharide (LPS)-induced macrophage inflammatory response. PolyP up-regulated LPS-induced production of the inflammatory cytokines, such as tumor necrosis factor α, interleukin-1β, and interleukin-6, in macrophages, and the effect was polyP dose- and chain length-dependent. However, orthophosphate did not exhibit this effect. PolyP enhanced the LPS-induced intracellular macrophage inflammatory signals. Affinity analysis revealed that polyP interacts with LPS, inducing formation of small micelles, and the polyP-LPS complex enhanced the binding affinity of LPS to Toll-like receptor 4 (TLR4) on macrophages. These results suggest that inorganic polyP plays a critical role in promoting inflammatory response by enhancing the interaction between LPS and TLR4 in macrophages. [ABSTRACT FROM AUTHOR]

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. β2-Microglobulin (β2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of β2m in patients are observed at neutral pH, formation of β2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that β2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration-dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the β2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses. [ABSTRACT FROM AUTHOR]

We have developed an experimental apparatus named KOTOBUKI-1 for use in the coherent x-ray diffraction imaging experiments of frozen-hydrated non-crystalline particles at cryogenic temperature. The apparatus allows us to collect diffraction data for frozen-hydrated specimens at 66 K and provides an experimental environment to easily transfer frozen-hydrated specimens from liquid nitrogen storage to the specimen stage for x-ray exposure. Since 2012, the apparatus has been used in the single-shot diffraction data collection of non-crystalline biological cells and cellular components with dimensions from micrometer to submicrometer using x-ray free electron lasers at SACLA. Here we report on the performance of the KOTOBUKI-1 diffraction apparatus and some structure analyses of biological cells and cellular components. Based on the present results, we also discuss the future developments of diffraction apparatus for more efficient data collection. [ABSTRACT FROM AUTHOR]