Cardiorespiratory coordination reveals training-specific physiological adaptations.

PURPOSE: To compare the effects of high-intensity interval training (HIIT) and moderate-intensity training (CONT), matched for total work, on cardiorespiratory coordination and aerobic fitness. METHODS: This is a two-arm parallel group single-blind randomised study. Twenty adults were assigned to 6 weeks of HIIT or volume-matched CONT. Participants completed a progressive maximal cycling test before and after the training period. Principal component (PC) analysis was performed on the series of cardiorespiratory variables to evaluate dimensionality of cardiorespiratory coordination, before and after lactate turnpoint. PC1 eigenvalues were compared. RESULTS: Both HIIT and CONT improved aerobic fitness (main effects of time, p < 0.001, [Formula: see text] ≥ 0.580), with no differences between groups. CONT decreased the number of PCs from two to one at intensities both below and above the lactate turnpoint; PC1 eigenvalues increased after CONT both below (Z = 2.08; p = 0.04; d = 0.94) and above the lactate turnpoint (Z = 2.10; p = 0.04; d = 1.37). HIIT decreased the number of PCs from two to one after the lactate turnpoint only; PC1 eigenvalues increased after HIIT above the lactate turnpoint (Z = 2.31; p = 0.02; d = 0.42). CONCLUSIONS: Although CONT and HIIT improved aerobic fitness to a similar extent, there were different patterns of change for cardiorespiratory coordination. These changes appear training-intensity specific and could be sensitive to investigate the individual response to endurance training.

Fucolipid metabolism as a function of cell population density in normal and murine sarcoma virus-transformed rat cells.

The incorporation of isotopically labeled fucose into the lipids of normal and murine sarcoma virus-transformed rat cells as a function of cell population density was examined. When normal cells were seeded at low cell density, the levels of the major fucolipids, i.e., fucolipids III and IV, were substantially reduced, but then they increased as the cells approached confluency. This variation in synthesis of fucolipids III and IV appeared to be primarily related to cell density and not to cell growth. Chase experiments revealed that the reduced level of fucolipids III and IV in sparse normal cells is due to decreased synthesis rather than to increased catabolism. In contrast to the observations with normal rat cells, the high level of fucolipid III and the low level of fucolipid IV in murine sarcoma virus-transformed rat cells was shown to be independent of cell population density.

Osmo-stress-induced changes in neutral trehalase activity of the fission yeast Schizosaccharomyces pombe.

Exposure of repressed growing cultures of Schizosaccharomyces pombe to various extracellular concentrations of NaCl, sorbitol or glycerol resulted in a reversible increase in neutral trehalase activity which was maintained while the cells were in the presence of high environmental osmolarity. Treatment of osmo-stress-induced trehalase by phosphatase lead to a decreased activity indicating that the active enzyme is phosphorylated. The stress response following the osmotic shock required protein synthesis and was independent of the cAMP-dependent protein kinase pathway. Cells disrupted for wis] or phh1 (identical to sty1 and spc1), which encode members of the mitogen-activated protein kinase (MAPK) cascade, showed that the osmo-stress-induced increase in trehalase markedly diminished. In contrast, the heat shock-induced increase in trehalase remained unchanged in these cells. Taken together, the data suggest that the elevation of trehalase activity in Schiz. pombe under conditions of high osmolarity is due to de novo synthesis of the enzyme and that this process is modulated through a MAPK signal transduction pathway as part of the physiological response to the osmotic stress. The wisl-phhl MAPK cascade, however, does not appear to form part of the mechanism underlaying the increase in trehalase after heat stress.

Trehalose-6P synthase is essential for trehalase activation triggered by glucose, nitrogen source or heat shock, but not by osmostress, in Schizosaccharomyces pombe.

Cells of Schizosaccharomyces pombe disrupted in the tps1+ gene, which encodes trehalose-6P synthase, were unable to increase trehalase activity in response to the addition of glucose or nitrogen source. Moreover, in contrast to normal cells, Deltatps1 cells did not increase trehalase activity by heat shock. Overexpression of tps1+ in cells devoid of trehalose-6P synthase restored the ability to increase trehalase after addition of nutrients or by heat shock. In glucose-repressed cells, which are normally refractory to the activation of trehalase by glucose, overexpression of tps1+ enabled the cells to increase trehalase activity upon addition of the sugar. Northern hybridisations were used to determine the level of mRNA for trehalase in normal and Deltatps1 cells. Transcription for trehalase was not significantly altered upon addition of glucose or nitrogen source, but increased markedly in heat-shocked cells even though trehalase activity remained unchanged in Deltatps1 cells. These findings provide evidence for a role of trehalose-6P synthase in the signalling pathway causing post-transcriptional activation of neutral trehalase induced by nutrients or heat shock. However, trehalase increased in Deltatps1 cells under hypertonic conditions suggesting the existence in Schiz. pombe of a distinct regulatory mechanism for enhancement of trehalase, specifically triggered by osmostress.

Analysis of the ntp1+ gene, encoding neutral trehalase in the fission yeast Schizosaccharomyces pombe.

We have cloned and sequenced the ntp1+ gene that codes for neutral trehalase in the fission yeast Schizosaccharomyces pombe. The ntp1+ gene product (Ntp1p) showed a 45-55% identity with neutral trehalases from other yeasts at the amino acid sequence level. However, in clear contrast to other neutral yeast trehalases so far characterized (which show two cAMP phospho-sites), only one consensus site for cAMP-dependent protein phosphorylation was found in Ntp1p. Northern blot hybridization experiments demonstrated that the Wis-Phh1/Sty1 MAP kinase cascade regulates ntp1+ expression during osmostress.

Activation of neutral trehalase by glucose and nitrogen source in Schizosaccharomyces pombe strains deficient in cAMP-dependent protein kinase activity.

Schizosaccharomyces pombe cells carrying a disruption in the PKA1 gene, that encodes the catalytic subunit of cAMP-dependent protein kinase (PKA), lacked the glucose- and nitrogen-source-induced activation of trehalase at stationary-phase but rised trehalase activity in response to these compounds during the exponential phase of growth. Treatment by phosphatase of either glucose- or nitrogen-source-activated trehalase resulted in trehalase deactivation suggesting that phosphorylation of the enzyme protein occurs during activation. These data indicate that in growing cells of this yeast the mechanism responsible for the activation of trehalase can be independent of interactions with free catalytic subunits of PKA and related to a signaling pathway involving a type of protein kinase different from PKA.

Accumulation of trehalose by overexpression of tps1, coding for trehalose-6-phosphate synthase, causes increased resistance to multiple stresses in the fission yeast schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.

Presence of two trehalose-6-phosphate synthase enzymes in Candida utilis.

Total trehalose-6-phosphate synthase activity decreased in cell extracts from Candida utilis under conditions inducing activation of the regulatory trehalase by protein kinase catalysed phosphorylation. The synthase activity was reactivated by treatment with alkaline phosphatase revealing the presence of an enzyme whose activity is inactivated by reversible phosphorylation. The occurrence in the trehalose-6-phosphate synthase complex of a second synthase enzyme whose activity is not controlled by phosphorylation and dephosphorylation was demonstrated following gel filtration of cell extracts. The activity of the isolated enzymes was differently modified in vitro by the presence of alkaline phosphatase, ATP, glucose or protein kinase.

Proteolytic activation of alpha,alpha-trehalose 6-phosphate synthase in Candida utilis.

Total trehalose 6-phosphate synthase activity increased in cell-free extracts from Candida utilis following short-term preincubation of the enzyme samples at 37 degrees C. This endogenous activation was prevented by the inhibitors of serine-type proteases, phenylmethylsulfonyl fluoride, antipain or chymostatin, but not by other protease inhibitors such as pepstatin. Fractionation of the cell extracts by Sephadex G-200 gel filtration revealed that the activity of one of the two synthase enzymes present in these cells was enhanced after the activation treatment. These observations indicate the existence of a proteolytically activatable enzyme form in the trehalose 6-phosphate synthase complex of this yeast in addition to the previously characterized enzyme, whose activity appears to be inactivated by reversible phosphorylation.

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger.

Resting cells of the fission yeast Schizosaccharomyces pombe, suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen tannophilus, the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.

Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe.

Spores from Schizosaccharomyces pombe contain neutral and acid trehalases. When spores from strains disrupted for ntp1(+), which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores. Further outgrowth was also reduced. Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination. Inhibition by phloridzin of the sporulation-specific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores. These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process.

Presence of non-suppressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations.

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.

Solubilization and characterization of a cell wall-bound trehalase from ascospores of the fission yeast Schizosaccharomyces pombe.

The genome of the fission yeast Schizosaccharomyces pombe lacks sequence homologs to ath1 genes coding for acid trehalases in other yeasts or filamentous fungi. However, acid trehalase activity is present at the spore stage in the life cycle of the fission yeast. The enzyme responsible for this activity behaves as a surface enzyme covalently linked to the spore cell walls in both wild-type and ntp1 mutant strains devoid of neutral trehalase. Lytic treatment of particulated cell wall fractions allowed the solubilization of the enzyme into an active form. We have characterized this soluble enzyme and found that its kinetic parameters, optimum pH and temperature, thermal denaturation and salt responses are closely similar to other conventional acid trehalases. Hence, this rather unusual enzyme can be recognized as acid trehalase by its biochemical properties although it does not share genetic homology with other known acid trehalases. The potential role of such acid trehalase in the mobilization of trehalose is discussed.

Loss of cell-population-density-dependent incorporation of fucose into the lipid fraction of cultured human tumour cells.

The incorporation of radioactively labelled fucose into the lipid fraction of cultured normal human cells and several human tumour-cell lines was examined as a function of the cell population density. Normal cells exhibited a density-dependent pattern of incorporation, whereas in tumour cells the radioactivity incorporated was independent of the cell population density. An exception was found among the tumour cells, which suggests a possible correlation between the loss of this marker and the ability to produce tumours.

Differential location of regulatory and nonregulatory trehalases in Candida utilis cells.

The isolation of vacuoles by density gradient centrifugation of protoplast lysates from Candida utilis cells showed a high specific activity for nonregulatory trehalase in vacuoles whereas the regulatory trehalase activatable by phosphorylation behaves as a cytoplasmic enzyme. The vacuolar trehalase is a glycoprotein that can be precipitated by Con A-Sepharose. Treatment of this enzyme with endo H reduced its reactivity with the lectin without loss of enzyme activity and decreased its apparent molecular weight by gel filtration.

Inhibition by alpha factor of the glucose-induced activation of regulatory trehalase in Saccharomyces cerevisiae.

The enzyme activity of the regulatory trehalase (alpha, alpha-trehalose glycohydrolase; EC 3.2.1.28) in stationary-phase cells of Saccharomyces cerevisiae increased upon addition of glucose to cell suspensions. Such increase was temporarily retarded in the presence of alpha factor in the medium. The transient inhibition required the joined action of the pheromone-like factor and the protease inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone. The inhibition of the glucose-induced activation of trehalase by alpha factor lends support to the involvement of adenosine 3',5'-cyclic monophosphate in the enzyme activation in vivo.

Comparative study of two trehalases from Candida utilis.

Candida utilis ATCC 60459 contains two intracellular trehalase enzymes clearly distinguishable by molecular weight, behaviour in ion-exchange chromatography and kinetic properties. The high molecular weight trehalase (500 kDa trehalase) is specifically inhibited by acetate and accounts for less than 30% of the total trehalase activity found in cell extracts. The smaller trehalase (280 kDa trehalase) exists mostly as a cryptic enzyme whose activity can be postranslationally activated by cAMP-dependent phosphorylation. The enzyme activity of the 280 kDa trehalase is strongly inhibited by Zn2+ and markedly enhanced in the presence of Ca2+ and Mn2+. The activation by these cations, contrariwise to that induced by ATP and cAMP, does not imply a covalent modification of the 280 kDa enzyme. Several parameters have been determined for both enzymes. The 280 kDa enzyme has the properties shown by the so-called regulatory trehalases whereas the 500 kDa enzyme presents characteristics of a nonregulatory type of trehalase.

Cyclic AMP signalling pathway and trehalase activation in the fission yeast Schizosaccharomyces pombe.

The response of derepressed cells of Schizosaccharomyces pombe to the addition of glucose included a marked and reversible activation of neutral trehalase that was not produced in repressed cells. The protein synthesis inhibitor cycloheximide, the protonophore 2,4-dinitrophenol or the uncoupler sodium azide also enhanced trehalase activity in derepressed cells provided glucose was present in the incubation assays. However, only 2,4-dinitrophenol or cycloheximide was able to induce trehalase activation in repressed cells. Stimulation of trehalase by these compounds was preceded in all cases by a rapid increase in adenosine 3'-5'-cyclic monophosphate (cAMP) content. Since exogenous cAMP can activate trehalase both in repressed and derepressed growing cells, the results provide evidence for the existence of an induced cAMP signalling pathway in the fission yeast with several entries for trehalase activation. The correlation between cAMP increase and trehalase activation was not maintained when the enzyme was heat-shock-activated, supporting the concept that trehalase activity can be also enhanced in cells by another mechanism in which cAMP does not act as second messenger.

On the role of trehalose in yeast cells subjected to hypersomotic shock.

Plate counts on both high and low water activity (aw) media were performed during the growth of Candida utilis in batch culture. The results revealed a marked discrepancy between the counts on the two media in the logarithmic phase. The discrepancy almost disappeared in stationary phase revealing a higher resistance of these cells, as compared to growing cells, to the severe dehydration imposed by the hyperosmotic shock upon transfer to low aw medium. Since the two types of cells differ in the level of endogenous trehalose the relation between plating discrepancy and trehalose content of the cells was investigated. Treatments that changed the intracellular trehalose concentration did not modify the plate counts on low aw medium. It was therefore concluded that the amount of trehalose into the cells is not the only factor conferring resistance against the hyperosmotic shock. Glycerol content did not correlate with resistance to the water stress either. Congruent with the former conclusion, other yeast species (Sporobolomyces salmonicolor, Schizosaccharomyces pombe) showed no correlation between changes in the trehalose content and susceptibility or resistance to the osmotic stress in low aw medium.

Sensitivity of fructose-1,6-bisphosphatase to glucose and cyclic AMP in the fission yeast Schizosaccharomyces pombe.

Stationary phase cells from resting cultures of Schizosaccharomyces pombe, suspended in buffer, did not decrease fructose-1,6-bisphosphatase (FbPase) activity upon addition of glucose to the cell suspension. In contrast, the addition of glucose to cells from derepressed growing cultures resulted in a 4- to 5-fold reduction in FbPase activity within minutes after the addition of the sugar. This response was independent of protein synthesis, was accompanied by a rise in the cyclic AMP (cAMP) level and was concomitant with an increase in the activity of neutral trehalase. The addition of exogenous cAMP to these cells provoked a decrease in FbPase similar to that induced by glucose. The occurrence of a glucose-induced cAMP signal was not sufficient to trigger the decrease in FbPase activity, which appeared to require additional transduction elements not active in growth-arrested cells. In assays performed in vitro it was found that the enzyme activity was strongly inhibited by fructose-2,6-bisphosphate.