Plasma Gelsolin Depletion and Circulating Actin in Sepsis—A Pilot Study

Background

Depletion of the circulating actin-binding protein, plasma gelsolin (pGSN) has been described in septic patients and animals. We hypothesized that the extent of pGSN reduction correlates with outcomes of septic patients and that circulating actin is a manifestation of sepsis.

Methodology/Principal Findings

We assayed pGSN in plasma samples from non-surgical septic patients identified from a pre-existing database which prospectively enrolled patients admitted to adult intensive care units at an academic hospital. We identified 21 non-surgical septic patients for the study. Actinemia was detected in 17 of the 21 patients, suggesting actin released into circulation from injured tissues is a manifestation of sepsis. Furthermore, we documented the depletion of pGSN in human clinical sepsis, and that the survivors had significantly higher pGSN levels than the non-survivors (163±47 mg/L vs. 89±48 mg/L, p = 0.01). pGSN levels were more strongly predictive of 28-day mortality than APACHE III scores. For every quartile reduction in pGSN, the odds of death increased 3.4-fold.

Conclusion

We conclude that circulating actin and pGSN deficiency are associated with early sepsis. The degree of pGSN deficiency correlates with sepsis mortality. Reversing pGSN deficiency may be an effective treatment for sepsis.     

Salivary alpha amylase and salivary cortisol response to fluid consumption in exercising athletes

The objective of the study was to examine salivary biomarker response to fluid consumption in exercising athletes. Exercise induces stress on the body and salivary alpha amylase (sAA) and salivary cortisol are useful biomarkers for activity in the sympathoadrenal medullary system and the hypothalamic pituitary adrenal axis which are involved in the stress response. Fifteen college students were given 150 ml and 500 ml of water on different days and blinded to fluid condition. The exercise protocol was identical for both fluid conditions using absolute exercise intensities ranging from moderate to high. Saliva was collected prior to exercise, post moderate and post high intensities and analyzed by Salimetrics assays. Exercise was significant for sAA with values different between pre-exercise (85 ± 10 U · ml⁻¹) and high intensity (284 ± 30 U · ml⁻¹) as well as between moderate intensity (204 ± 32 U · ml⁻¹) and high intensity. There was no difference in sAA values between fluid conditions at either intensity. Exercise intensity and fluid condition were each significant for cortisol. Cortisol values were different between pre-exercise (0.30 ± 0.03 ug · dL⁻¹) and high intensity (0.45 ± 0.05 ug · dL⁻¹) as well as between moderate intensity (0.33 ± 0.04 ug · dL⁻¹) and high intensity. Moderate exercise intensity cortisol was lower in the 500 ml condition (0.33 ± 0.03 ug · dL⁻¹) compared with the 150 ml condition (0.38 ± 0.03 ug · dL⁻¹). This altered physiological response due to fluid consumption could influence sport performance and should be considered. In addition, future sport and exercise studies should control for fluid consumption.     

Cryoprotectants and Extreme Freeze Tolerance in a Subarctic Population of the Wood Frog

Wood frogs (Rana sylvatica) exhibit marked geographic variation in freeze tolerance, with subarctic populations tolerating experimental freezing to temperatures at least 10-13 degrees Celsius below the lethal limits for conspecifics from more temperate locales. We determined how seasonal responses enhance the cryoprotectant system in these northern frogs, and also investigated their physiological responses to somatic freezing at extreme temperatures. Alaskan frogs collected in late summer had plasma urea levels near 10 μmol ml^-1, but this level rose during preparation for winter to 85.5 ± 2.9 μmol ml^-1 (mean ± SEM) in frogs that remained fully hydrated, and to 186.9 ± 12.4 μmol ml^-1 in frogs held under a restricted moisture regime. An osmolality gap indicated that the plasma of winter-conditioned frogs contained an as yet unidentified osmolyte(s) that contributed about 75 mOsmol kg^-1 to total osmotic pressure. Experimental freezing to –8°C, either directly or following three cycles of freezing/thawing between –4 and 0°C, or –16°C increased the liver’s synthesis of glucose and, to a lesser extent, urea. Concomitantly, organs shed up to one-half (skeletal muscle) or two-thirds (liver) of their water, with cryoprotectant in the remaining fluid reaching concentrations as high as 0.2 and 2.1 M, respectively. Freeze/thaw cycling, which was readily survived by winter-conditioned frogs, greatly increased hepatic glycogenolysis and delivery of glucose (but not urea) to skeletal muscle. We conclude that cryoprotectant accrual in anticipation of and in response to freezing have been greatly enhanced and contribute to extreme freeze tolerance in northern R. sylvatica.     

Penicillium ochrochloron MTCC 517 chitinase: An effective tool in commercial enzyme cocktail for production and regeneration of protoplasts from various fungi

Penicillium ochrochloron MTCC 517 is a potent producer of chitinolytic enzymes. Novozyme 234, traditional enzyme cocktail for protoplast generation is not available in the market. So, new enzyme cocktail is prepared for protoplast formation from various filamentous fungi which consists of 5 mg ml⁻¹ lysing enzymes from Trichoderma harzianum, 0.06 mg ml⁻¹ β-glucuronidase from Helix pomatia and 1 mg ml⁻¹P. ochrochloron chitinase. The greatest number of protoplasts could be produced from most of the fungi in 0.8 M sorbitol and by incubation for about 2 h at 37 °C, but the number was decreased by incubation for more than 3 h. About twice as many protoplasts were produced from different species of fungi by involvement of P. ochrochloron chitinase than with combined commercial enzymes.     

PRE-EXERCISE ARGININE SUPPLEMENTATION INCREASES TIME TO EXHAUSTION IN ELITE MALE WRESTLERS

Dietary supplements containing arginine are among the most popular ergogenics intended to enhance strength, power and muscle recovery associated with both anaerobic and aerobic exercise. The aim of the present study was to evaluate the possible effect of pre-exercise acute intake of arginine on performance and exercise metabolism during incremental exhaustive exercise in elite male wrestlers. Nine volunteer elite male wrestlers (24.7±3.8 years) participated in this study. The test-retest protocol was used on the same subjects. The study was conducted using a cross-over design. A single dose of arginine (1.5 g · 10 kg^-1 body weight) or placebo was given to the subjects after 12 hours fasting (during the night) for both test and retest. Subjects were allowed to drink water but not allowed to eat anything between arginine or placebo ingestion and the exercise protocol. An incremental exercise protocol was applied and oxygen consumption was measured during the exercise. Heart rate and plasma lactate levels were measured during the exercise and recovery. Results showed that in the same working loads there was no significant difference for the mean lactate levels and no difference in maximum oxygen consumption (arginine 52.47±4.01 mL · kg^-1 · min^-1, placebo 52.07±5.21 mL · kg^-1 · min^-1) or in maximum heart rates (arginine 181.09±13.57 bpm, placebo 185.89±7.38 bpm) between arginine and placebo trials. Time to exhaustion was longer with arginine supplementation (1386.8±69.8 s) compared to placebo (1313±90.8 s) (p < 0.05). These results suggest that L-arginine supplementation can have beneficial effects on exercise performance in elite male wrestlers but cannot explain the metabolic pathways which are responsible from these effects.     

Culturable Heavy Metal-Resistant and Plant Growth Promoting Bacteria in V-Ti Magnetite Mine Tailing Soil from Panzhihua,China

To provide a basis for using indigenous bacteria for bioremediation of heavy metal contaminated soil, the heavy metal resistance and plant growth-promoting activity of 136 isolates from V-Ti magnetite mine tailing soil were systematically analyzed. Among the 13 identified bacterial genera, the most abundant genus was Bacillus (79 isolates) out of which 32 represented B. subtilis and 14 B. pumilus, followed by Rhizobium sp. (29 isolates) and Ochrobactrum intermedium (13 isolates). Altogether 93 isolates tolerated the highest concentration (1000 mg kg⁻¹) of at least one of the six tested heavy metals. Five strains were tolerant against all the tested heavy metals, 71 strains tolerated 1,000 mg kg⁻¹ cadmium whereas only one strain tolerated 1,000 mg kg⁻¹ cobalt. Altogether 67% of the bacteria produced indoleacetic acid (IAA), a plant growth-promoting phytohormone. The concentration of IAA produced by 53 isolates was higher than 20 µg ml⁻¹. In total 21% of the bacteria produced siderophore (5.50–167.67 µg ml⁻¹) with two Bacillus sp. producing more than 100 µg ml⁻¹. Eighteen isolates produced both IAA and siderophore. The results suggested that the indigenous bacteria in the soil have beneficial characteristics for remediating the contaminated mine tailing soil.     

The influence of environmental temperature on appetite-related hormonal responses

Background

Acute exercise in the heat has been shown to reduce appetite. However, the influence of exercise in the cold on appetite regulation remains unclear. The aim of this study was to compare exercise-induced appetite regulation under three different environmental temperatures.

Methods

Eleven male participants completed three experimental trials on the following separate days: exercise in the heat (36°C), exercise at neutral temperature (24°C), and exercise in the cold (12°C). The exercise trials consisted of pedaling exercises for 30 min at 65% of maximal oxygen uptake (VO_2max). Blood samples were collected repeatedly to determine plasma ghrelin, peptide YY (PYY) and other hormonal concentrations. Subjective feelings of hunger and tympanic temperature were also monitored.

Results

Tympanic temperature was significantly higher in the 36°C trial than that of the other two trials (P < 0.05). The subjective feelings of hunger in the 36°C and 24°C trials were significantly lower than those in the 12°C trial (P < 0.05). Plasma ghrelin concentration decreased significantly with exercise in all conditions (P < 0.05), and the responses were not significantly different among the three conditions. Plasma PYY concentration increased significantly after the exercise in the 24°C trial only (P < 0.05), with no significant difference among the three trials.

Conclusions

These results suggest that exposure to hot or cold temperatures during exercise did not affect exercise-induced plasma ghrelin and PYY responses. However, the exercise-induced reduction of subjective hunger was significantly attenuated in a cold environment.     

Sequential Exercise in Triathletes: Variations in GH and Water Loss

Growth hormone (GH) may stimulate water loss during exercise by activating sweating. This study investigated GH secretion and water loss during sequential cycling and running, taking postural changes into account. The two exercise segments had similar durations and were performed at the same relative intensity to determine their respective contributions to water loss and the plasma volume variation noted in such trials. Eight elite triathletes first performed an incremental cycle test to assess maximal oxygen consumption. Then, the triathletes performed one of two trials in randomized order: constant submaximal cycling followed by treadmill running (C₁-R₂) or an inversed succession of running followed by cycling (R₁-C₂). Each segment of both trials was performed for 20 minutes at ∼75% of maximal oxygen consumption. The second trial, reversing the segment order of the first trial, took place two weeks later. During cycling, the triathletes used their own bicycles equipped with a profiled handlebar. Blood sampling (for GH concentrations, plasma viscosity and plasma volume variation) was conducted at rest and after each segment while water loss was estimated from the post- and pre-measures. GH increases were significantly lower in R₂ than C₂ (72.2±50.1 vs. 164.0±157 ng.ml⁻¹.min⁻¹, respectively; P<0.05). Water loss was significantly lower after C₁-R₂ than R₁-C₂ (1105±163 and 1235±153 ml, respectively; P<0.05). Plasma volume variation was significantly negative in C₁ and R₁ (−6.15±2.0 and −3.16±5.0%, respectively; P<0.05), not significant in C₂, and significantly positive for seven subjects in R₂ (4.05±3.1%). We concluded that the lower GH increases in R₂ may have contributed to the smaller reduction in plasma volume by reducing sweating. Moreover, this lower GH response could be explained by the postural change during the transition from cycling to running. We recommend to pay particular attention to their hydration status during R₁ which could limit a potential dehydration during C₂.     

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch,slow-twitch,and cardiac muscle

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s⁻¹) and super-relaxed states (SRX, 0.005 s⁻¹) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969–1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C–30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.     

Reduced susceptibility to eccentric exercise-induced muscle damage in resistance-trained men is not linked to resistance training-related neural adaptations

The purpose of this study was to examine the acute effects of maximal concentric vs. eccentric exercise on the isometric strength of the elbow flexor, as well as the biceps brachii muscle electromyographic (EMG) responses in resistance-trained (RT) vs. untrained (UT) men. Thirteen RT men (age: 24 ± 4 years; height: 180.2 ± 7.7 cm; body weight: 92.2 ± 16.9 kg) and twelve UT men (age: 23 ± 4 years; height: 179.2 ± 5.0 cm; body weight: 81.5 ± 8.6 kg) performed six sets of ten maximal concentric isokinetic (CON) or eccentric isokinetic (ECC) elbow flexion exercise in two separate visits. Before and after the exercise interventions, maximal voluntary contractions (MVCs) were performed for testing isometric strength. In addition, bipolar surface EMG signals were detected from the biceps brachii muscle during the strength testing. Both CON and ECC caused isometric strength to decrease, regardless of the training status. However, ECC caused greater isometric strength decline than CON did for the UT group (p = 0.006), but not for the RT group. Both EMG amplitude and mean frequency significantly decreased and increased, respectively, regardless of the training status and exercise intervention. Resistance-trained men are less susceptible to eccentric exercise-induced muscle damage, but this advantage is not likely linked to the chronic resistance training-induced neural adaptations.     

The anti-inflammatory effects of exercise training promote atherosclerotic plaque stabilization in apolipoprotein E knockout mice with diabetic atherosclerosis

Physical exercise is the cornerstone of cardiovascular disease treatment. The present study investigated whether exercise training affects atherosclerotic plaque composition through the modification of inflammatoryrelated pathways in apolipoprotein E knockout (apoE^−/−) mice with diabetic atherosclerosis. Forty-five male apoE^−/− mice were randomized into three equivalent (n=15) groups: control (CO), sedentary (SED), and exercise (EX). Diabetes was induced by streptozotocin administration. High-fat diet was administered to all groups for 12 weeks. Afterwards, CO mice were euthanatized, while the sedentary and exercise groups continued high-fat diet for 6 additional weeks. Exercising mice followed an exercise program on motorizedtreadmill (5 times/week, 60 min/session). Then, blood samples and atherosclerotic plaques in the aortic root were examined. A considerable (P<0.001) regression of the atherosclerotic lesions was observed in the exercise group (180.339±75.613×10³µm²) compared to the control (325.485±72.302×10³ µm²) and sedentary (340.188±159.108×10³µm²) groups. We found decreased macrophages, matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-8 and interleukin-6 (IL-6) concentrations (P<0.05) in the atherosclerotic plaques of the exercise group. Compared to both control and sedentary groups, exercise training significantly increased collagen (P<0.05), elastin (P<0.001), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) (P<0.001) content in the atherosclerotic plaques. Those effects paralleled with increased fibrous cap thickness and less internal elastic lamina ruptures after exercise training (P<0.05), while body-weight and lipid parameters did not significantly change. Plasma MMP-2 and MMP-3 concentrations in atherosclerotic tissues followed a similar trend. From our study we can conclude that exercise training reduces and stabilizes atherosclerotic lesions in apoE^−/− mice with diabetic atherosclerosis. A favorable modification of the inflammatory regulators seems to explain those beneficial effects.Key words: diabetes, atherosclerosis, exercise, matrix metalloproteinases, plaque stability.     

Therapeutic potential of plasma gelsolin administration in a rat model of sepsis

BackgroundGelsolin is an actin-binding protein found in the cytoplasm and in extracellular fluids including blood plasma. Plasma gelsolin concentration decreases after a wide range of injuries. We hypothesized that the repletion of gelsolin would limit inflammation and tissue injury in a rat model of sepsis using cecal ligation and double puncture (2CLP).MethodsHuman plasma gelsolin (pGSN, 10 mg in 1 ml saline) was administered once immediately following surgery, and control 2CLP (2CLP Alb) and sham animals were injected with 1 ml saline containing equimolar albumin. Treatments were administered intraperitoneally (IP), intravenously (IV), or subcutaneously (SC).ResultsGelsolin levels in the 2CLP Alb group were lower than in sham animals. Administration of pGSN increased levels when administered IV and SC, but not IP. Morbidity scores were significantly less severe in the 2CLP pGSN group than in the 2CLP Alb group when pGSN was administered IV and SC, but not IP. Furthermore, enzymatic activity indicative of tissue damage (lactate dehydrogenase and alanine transaminase) was significantly lower in 2CLP pGSN group when treated SC compared to 2CLP Alb group.ConclusionThese data provide further evidence that exogenous gelsolin can reduce morbidity from sepsis.     

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent K_m for starch was 0.8 mg ml⁻¹. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml⁻¹ (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml⁻¹ in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml⁻¹), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml⁻¹), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.     

Human Muscle Protein Synthetic Responses during Weight-Bearing and Non-Weight-Bearing Exercise: A Comparative Study of Exercise Modes and Recovery Nutrition

Effects of conventional endurance (CE) exercise and essential amino acid (EAA) supplementation on protein turnover are well described. Protein turnover responses to weighted endurance exercise (i.e., load carriage, LC) and EAA may differ from CE, because the mechanical forces and contractile properties of LC and CE likely differ. This study examined muscle protein synthesis (MPS) and whole-body protein turnover in response to LC and CE, with and without EAA supplementation, using stable isotope amino acid tracer infusions. Forty adults (mean ± SD, 22 ± 4 y, 80 ± 10 kg, VO_2peak 4.0 ± 0.5 L∙min^-1) were randomly assigned to perform 90 min, absolute intensity-matched (2.2 ± 0.1 VO₂ L∙m^-1) LC (performed on a treadmill wearing a vest equal to 30% of individual body mass, mean ± SD load carried 24 ± 3 kg) or CE (cycle ergometry performed at the same absolute VO₂ as LC) exercise, during which EAA (10 g EAA, 3.6 g leucine) or control (CON, non-nutritive) drinks were consumed. Mixed-muscle and myofibrillar MPS were higher during exercise for LC than CE (mode main effect, P < 0.05), independent of dietary treatment. EAA enhanced mixed-muscle and sarcoplasmic MPS during exercise, regardless of mode (drink main effect, P < 0.05). Mixed-muscle and sarcoplasmic MPS were higher in recovery for LC than CE (mode main effect, P < 0.05). No other differences or interactions (mode x drink) were observed. However, EAA attenuated whole-body protein breakdown, increased amino acid oxidation, and enhanced net protein balance in recovery compared to CON, regardless of exercise mode (P < 0.05). These data show that, although whole-body protein turnover responses to absolute VO₂-matched LC and CE are the same, LC elicited a greater muscle protein synthetic response than CE.     

Nuclear Magnetic Resonance Studies of Sodium Ions in Isolated Frog Muscle and Liver

Nuclear magnetic resonance (NMR) was used to determine Na⁺ complexing in muscle and liver (at 23°C) from bullfrogs (Rana catesbeiana) and to study the influence of temperature on Na⁺ complexing in muscle from leopard frogs (Rana pipiens). The Na⁺ complexed in muscle and liver was found to be 36.6 ± 4.6% and 66.1 ± 3.5% respectively. A temperature decrease from +34°C to -2°C results in a 20% decrease in the mobility of the free Na⁺ in the fresh muscle. This 20% decrease in mobility results in about 50% of the free Na⁺ at 34°C being complexed at the lower temperature.     

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca²⁺-dependent, but not completely reversible, as on removal of Ca²⁺ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca²⁺ in a micromolar range, with half maximum activation at about 3×10⁻⁶ M. The protein becomes inactivated when Ca²⁺ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2–4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.     

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml⁻¹. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g⁻¹ (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml⁻¹ in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 10⁸ to 6 × 10⁸ CFU ml⁻¹. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.     

Gelsolin Modulates Phospholipase C Activity In Vivo through Phospholipid Binding

Gelsolin and CapG are actin regulatory proteins that remodel the cytoskeleton in response to phosphatidylinositol 4,5-bisphosphate (PIP₂) and Ca²⁺ during agonist stimulation. A physiologically relevant rise in Ca²⁺ increases their affinity for PIP₂ and can promote significant interactions with PIP₂ in activated cells. This may impact divergent PIP₂- dependent signaling processes at the level of substrate availability. We found that CapG overexpression enhances PDGF-stimulated phospholipase C_γ (PLC_γ) activity (Sun, H.-q., K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. J. Cell Biol. 129:147–156). In this paper, we examined the ability of gelsolin and CapG to compete with another PLC for PIP₂ in live cells, in semiintact cells, and in vitro. We found that CapG and gelsolin overexpression profoundly inhibited bradykinin-stimulated PLC_β. Inhibition occurred at or after the G protein activation step because overexpression also reduced the response to direct G protein activation with NaF. Bradykinin responsiveness was restored after cytosolic proteins, including gelsolin, leaked out of the overexpressing cells. Conversely, exogenous gelsolin added to permeabilized cells inhibited response in a dose-dependent manner. The washout and addback experiments clearly establish that excess gelsolin is the primary cause of PLC inhibition in cells. In vitro experiments showed that gelsolin and CapG stimulated as well as inhibited PLC_β, and only gelsolin domains containing PIP₂-binding sites were effective. Inhibition was mitigated by increasing PIP₂ concentration in a manner consistent with competition between gelsolin and PLC_β for PIP₂. Gelsolin and CapG also had biphasic effects on tyrosine kinase– phosphorylated PLC_γ, although they inhibited PLC_γ less than PLC_β. Our findings indicate that as PIP₂ level and availability change during signaling, cross talk between PIP₂-regulated proteins provides a selective mechanism for positive as well as negative regulation of the signal transduction cascade.     

Twelve Weeks of Sprint Interval Training Improves Indices of Cardiometabolic Health Similar to Traditional Endurance Training despite a Five-Fold Lower Exercise Volume and Time Commitment

AimsWe investigated whether sprint interval training (SIT) was a time-efficient exercise strategy to improve insulin sensitivity and other indices of cardiometabolic health to the same extent as traditional moderate-intensity continuous training (MICT). SIT involved 1 minute of intense exercise within a 10-minute time commitment, whereas MICT involved 50 minutes of continuous exercise per session.MethodsSedentary men (27±8y; BMI = 26±6kg/m²) performed three weekly sessions of SIT (n = 9) or MICT (n = 10) for 12 weeks or served as non-training controls (n = 6). SIT involved 3x20-second ‘all-out’ cycle sprints (~500W) interspersed with 2 minutes of cycling at 50W, whereas MICT involved 45 minutes of continuous cycling at ~70% maximal heart rate (~110W). Both protocols involved a 2-minute warm-up and 3-minute cool-down at 50W.ResultsPeak oxygen uptake increased after training by 19% in both groups (SIT: 32±7 to 38±8; MICT: 34±6 to 40±8ml/kg/min; p<0.001 for both). Insulin sensitivity index (CS_I), determined by intravenous glucose tolerance tests performed before and 72 hours after training, increased similarly after SIT (4.9±2.5 to 7.5±4.7, p = 0.002) and MICT (5.0±3.3 to 6.7±5.0 x 10⁻⁴ min^-1 [μU/mL]^-1, p = 0.013) (p<0.05). Skeletal muscle mitochondrial content also increased similarly after SIT and MICT, as primarily reflected by the maximal activity of citrate synthase (CS; P<0.001). The corresponding changes in the control group were small for VO₂peak (p = 0.99), CS_I (p = 0.63) and CS (p = 0.97).ConclusionsTwelve weeks of brief intense interval exercise improved indices of cardiometabolic health to the same extent as traditional endurance training in sedentary men, despite a five-fold lower exercise volume and time commitment.     

Chlorine Dioxide for Reduction of Postharvest Pathogen Inoculum during Handling of Tree Fruits

Alternatives to hypochlorous acid and fungicides are needed for treatment of fruit and fruit-handling facilities. Chlorine dioxide was evaluated and found effective against common postharvest decay fungi and against filamentous fungi occurring on fruit packinghouse surfaces. In vitro tests with conidial or sporangiospore suspensions of Botrytis cinerea, Penicillium expansum, Mucor piriformis, and Cryptosporiopsis perennans demonstrated >99% spore mortality within 1 min when the fungi were exposed to aqueous chlorine dioxide at 3 or 5 μg · ml^-1. Longer exposure times were necessary to achieve similar spore mortalities with 1 μg · ml^-1. Of the fungi tested, B. cinerea and P. expansum were the least sensitive to ClO₂. In comparison with the number recovered from untreated control areas, the number of filamentous fungi recovered was significantly lower in swipe tests from hard surfaces such as belts and pads in a commercial apple and pear packinghouse after treatment of surfaces with a 14.0- to 18.0-μg · ml^-1 ClO₂ foam formulation. Chlorine dioxide has desirable properties as a sanitizing agent for postharvest decay management when residues of postharvest fungicides are not desired or allowed.