Significant, but limited collateral blood flow increases occur with prolonged training in rats with femoral artery occlusion.

This study examined the capacity of collateral dependent blood flow induced by a prolonged treadmill training program, as compared to a low collateral resistance model created by femoral artery to vein (A-V) shunt. Sprague-Dawley rats, with bilateral femoral artery occlusion were confined to cage activity (Sed, n=9) or trained by daily treadmill exercise (Tr, n=15; up to ≈350 min/d) for 15 weeks. Another set of animals received a femoral A-V anastomosis in one limb and treated with (n=4) or without VEGF(165) (n=9) infusion for 2 weeks. The contralateral side was used as control. Blood flow (BF) was measured with isotope labeled microspheres. Maximal calf muscle BF increased by 15 week training (up to 100±5.0 ml x min(-1) x 100g(-1) (p<0.05); 0.71±0.04 ml x min(-1) x 100g(-1) x mmHg(-1)), a response better (20-25%) than the less demanding training programs used previously. In contrast, femoral A-V shunt with VEGF(165) increased calf muscle conductance to 1.70±0.3 ml x min(-1) x 100 g(-1) x mmHg(-1) that is similar to blood flows observed in non-occluded rats during maximal running. Our data indicate that the collateral circuit development is related to the driving stimulus and that exercise training, does not provide a maximal stimulus for adaptation that is possible. Nonetheless, exercise training results in profound increases in exercise capacity associated with this enhanced collateral blood flow. Our results illustrate that vascular adaptations can be much greater when physiologically induced stimuli are enhanced at the time of therapeutic angiogenesis.

Expression of CD57 on CD8+ T lymphocytes of patients with Wegener's granulomatosis and microscopic polyangiitis: evidence for continuous activation of CD8+ cells.

OBJECTIVES: To gain insight into the immune pathogenesis of Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA), the prevalence of circulating CD8+ T lymphocytes expressing CD57 as a marker for previous activation was analyzed. METHODS: Receptor expression of CD57 was measured in CD8+ T cells of patients with active disease (n=5) by cytofluorometry and compared with expression in patients in remission (n=80) and in age-matched healthy donors (n=34). The results were compared to clinical parameters including severity and duration of the disease. RESULTS: CD8+CD57+ were detected in patients with WG and MPA and in healthy donors as well and increased considerably with age. Compared to age-matched healthy donors, the prevalence of CD8+CD57+ was increased in the younger patients (up to 40 y). In most patients a high percentage of CD8+CD57+ coincided with severe disease and multiple organ involvement, while low CD8+CD57+ percentage was seen in patients with limited disease or in patients in complete remission. In patients with smoldering disease, the percentage of CD8+CD57+ increased with time. High numbers of CD8+CD57+ correlated with low CD4:CD8 ratio. CONCLUSIONS: In patients with WG and MPA a population of CD8+CD57+ expand, identifying terminally differentiated CD8+ cells. The prevalence of CD57+ cells was related to the course of disease. So far, the function of CD57 on CD8+ cells is not understood. However, these cells might produce certain cytokines, which play a role in the pathogenesis of AAV. The data support the hypothesis that CD8+ T cells are activated in the context of primary vasculitides.

The extracellular polymer substance of Pseudomonas aeruginosa: too slippery for neutrophils to migrate on?

PURPOSE: Biofilm formation is increasingly recognized as the cause of persistent infections and there is evidence that P. aeruginosa organized into biofilms are quite resistant toward host defence mechanisms, particularly against an attack by polymorphonuclear neutrophils (PMN). Apparently, the migration of PMN through the biofilms is impaired, and thus the bactericidal activity remains highly localized. The aim of this study was to directly investigate the interaction of PMN with the biofilm and the extracted extracellular polymeric substance (EPS) of P. aeruginosa. MATERIAL AND METHODS: Chemotaxis and random migration of PMN through P. aeruginosa biofilms was tested, as was their migration through and along the EPS. RESULTS: We found that the EPS and mature biofilms, but not immature or developing ones, reduced the chemotactic migration of PMN. On EPS, rather than immobilize the cells, their random, spontaneous migration was enhanced. CONCLUSION: We propose that on EPS, the PMN lose their capacity to sense the direction and just slide over the EPS in a disoriented manner.

T lymphocytes in patients with primary vasculitis: expansion of CD8+ T cells with the propensity to activate polymorphonuclear neutrophils.

OBJECTIVES: To gain insight into the immune pathogenesis of primary ANCA-associated vasculitides, the prevalence of circulating T lymphocytes expressing CD11b as a marker for activation was analysed in patients with WG or microscopic polyangiitis. METHODS; Receptor expression and IFNgamma synthesis were measured in T cells of patients with active disease by cytofluorometry and compared with expression in patients in remission and in healthy donors. RESULTS: During active disease, a small but conspicuous population of CD8+CD28+CD11b+ was found which produced IFNgamma. In healthy donors and in patients in remission or undergoing immunosuppressive therapy, CD11b was exclusively associated with CD8+CD28- cells, the latter being more frequent in patients with long-lasting or severe disease. In vitro experiments confirmed that CD11b is up-regulated when T cells are activated. After multiple rounds of restimulation, the CD11b expression persists whereas CD28 expression is lost, compatible with the notion that CD8+CD28+CD11b+ represents a transient phenotype in the course of T-cell activation. The IFNgamma-producing T cells activated polymorphonuclear neutrophils (PMN) to express MHC class II, thus generating the same PMN phenotype as in patients with active ANCA-associated vasculitis. A similar PMN phenotype could be generated by cultivation with supernatants of activated T cells or by IFNgamma alone, but not by antibodies to proteinase 3. CONCLUSIONS: In active primary vasculitis, a small population of CD8+ T cells, identified by the expression of CD11b, expands, producing IFNgamma. These T cells could activate PMN, thus generating a long-living and potentially destructive PMN phenotype.

Training-induced vascular adaptations to ischemic muscle.

Peripheral arterial insufficiency is a progressive degenerative disease associated with an increased morbidity and mortality. It decreases exercise tolerance and often presents with symptoms of intermittent claudication. Enhanced physical activity is one of the most effective means of improving the life of affected patients. While this occurs for a variety of reasons, vascular remodeling can be an important means for improved oxygen exchange and blood flow delivery. Relevant exercise-induced signals stimulate angiogenesis, within the active muscle (e.g. hypoxia), and arteriogenesis (enlargement of pre-existing vessels via increased shear stress) to increase oxygen exchange and blood flow capacity, respectively. Evidence from pre-clinical studies shows that the increase in collateral blood flow observed with exercise progresses over time of training, is accompanied by significant enlargement of isolated collateral vessels, and enhances the responses observed with angiogenic growth factors (e.g. VEGF, FGF-2). Thus, enhanced physical activity can be an effective means of enlarging the structure and function of the collateral circuit. Interestingly, disrupting normal NO production (via L-NAME) eliminates this increase in collateral blood flow induced by training, but does not disturb the increase in muscle capillarity within the active muscle. Similarly, inhibiting VEGF receptor kinase activity eliminates the increase in collateral-dependent blood flow, and lessens, but does not eliminate, angiogenesis within the calf muscle. These findings illustrate distinctions between the processes influencing angiogenesis and arteriogenesis. Further, sympathetic modulation of the collateral circuit does not eliminate the increase in collateral circuit conductance induced by exercise training. These findings indicate that structural enlargement of the collateral vessels is essential to realize the increase in collateral-dependent blood flow capacity caused by exercise training. This raises the potential that meaningful vascular remodeling can occur in patients with intermittent claudication who actively participate in exercise training.

Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils.

AIM: To establish a rapid, improved soil environmental DNA extraction and purification protocol. METHODS AND RESULTS: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. CONCLUSIONS: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.

A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi.

A new, rapid method for evaluation of lipid peroxidation promoting (pro-oxidant) activity in cultures of wood-decaying fungi was developed. The method is based on measurement of the rate of oxygen consumption in the reaction of linoleic acid peroxidation initiated by fungal culture filtrates. The liquid cultures of the white-rot fungi Bjerkandera adusta and Phanerochaete chrysosporium grown on wheat straw-containing glucose-peptone-corn steep liquor medium possessed significant levels of the pro-oxidant activity. Other white-rot fungi producing manganese peroxidase (MnP) were also found to show the activity. MnP demonstrated a crucial role as the major pro-oxidant agent in the fungal cultures. The total pro-oxidant activity may be considered as net result of the peroxidation by MnP and the inhibition by antioxidant compounds present in the fungal culture fluids.

Evaluation of yeast strains as possible agents for trace enrichment of metal ions in aquatic environments.

Sorption properties of six yeast strains were evaluated for trace enrichment of metal ions; Cd(2+), Cr(3+), Cr(6+), Cu(2+), Pb(2+), and Zn(2+) from aqueous environments. Metal concentration was determined by flame atomic absorption spectrometry (FAAS). The results showed that trace enrichment of the metals under study with yeast, was dependent on the pH and available metal ions. Enrichment time of 30min gave an optimum metal uptake. The presence of Na(+), K(+), and Ca(2+) suppressed the uptake of Pb by less than 5%, but suppressed the uptake of Zn by between 15 and 25%. Mg(2+), Cu(+), Cu(2+), Cr(3+) Cr(6+), Cd(2+), and Zn(2+) suppressed the uptake of Pb by between 25 and 35%, and that of Zn by between 15 and 25%. For both Pb and Zn, Cd had the highest suppression of 35 and 30%, respectively for baker's yeast (Saccharomyces cerevisiae). Baker's yeast achieved enrichment factors (EF) of 23, 4, 100, and 1 for dam water, stream water, treated wastewater, and industrial effluent samples for Cu, Pb, Zn, and Cr, respectively. The recoveries of optimised Cd and Cr samples spiked with 2mugml(-1) of the metal could reach up to 90%, but never exceeded 66% for 10mugml(-1) samples. For Cu and Pb, the recoveries generally increased independent of concentration, however they were not as high as those for Zn, which exceeded 90% for all the samples spiked with 10mugml(-1) of the metal. S. cerevisiae PR 61/3 had the highest EF for Cr as compared to the other yeast strains. S. cerevisiae PRI 60/78 was the only yeast strain which was able to enrich Cd in all the samples. Baker's yeast had the highest EFs for Cu and Zn as compared to the other yeast strains without pH adjustment of the water samples. Candida tropicalis attained the highest EFs for Pb as compared to the other yeast strains. The results indicate that all the yeast strains used had a high affinity for Zn based on the EF values achieved. The results from these studies demonstrate that yeast is a viable trace metal enrichment agent that can be used freely suspended in solution to enrich metal ions at relatively low concentrations. This has ramifications on the traditional methods of sampling, sample collection, and transportation from remote sampling sites.

Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis.

An extracellular alkaline protease produced by Bacillus licheniformis AP-1 was purified 76-fold, yielding a single 28 kDa band on SDS-PAGE. It was optimally active at pH 11 and at 60 degrees C (assayed over 10 min). The protease was completely inhibited by phenylmethylsulfonyl fluoride and diodopropyl fluorophosphate, with little increase upon Ca2+ and Mg2+ addition.

Cloning and over-expression of an alkaline protease from Bacillus licheniformis.

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.

MRI evaluation of topical heat and static stretching as therapeutic modalities for the treatment of eccentric exercise-induced muscle damage.

The aim of this study was to monitor the effects of topical heat and/or static stretch treatments on the recovery of muscle damage by eccentric exercise. For this purpose, 32 untrained male subjects performed intense eccentric knee extension exercise, followed by 2 weeks of treatment (heat, stretch, heat plus stretch) or no treatment (control, n=8/group). Isometric strength testing, pain ratings, and multi-echo magnetic resonance imaging of the thigh were performed before and at 2, 3, 4, 8, and 15 days following the exercise. Increased T2 relaxation time, muscle swelling, pain ratings, and strength loss confirmed significant muscle damage during the post-exercise period. Pain ratings and muscle volume recovered to baseline by 15 days, although muscle strength remained lower [77 (4) vs. 95 (3) kg pre-exercise, mean (SE)] and T2 values higher [32.2 (0.8) vs. 28.6 (0.2) ms pre-exercise]. Our results indicate that heat and/or static stretching does not consistently reduce soreness, swelling or muscle damage. The practical implication of our findings is that clinicians should be aware that prescribing heat and/or static stretching following intense eccentric or unaccustomed exercise will not enhance the recovery of damaged muscles.

Arteriogenesis: role of nitric oxide.

Arteriogenesis is an important process for adapting the pre-existing circuit of vessels into functional collateral conduits for delivery of oxygen enriched blood to tissue distal to occlusion of a large, peripheral conduit artery. Recent evidence has shown that arteriogenesis is regulated by nitric oxide (NO), angiogenic factors and shear stress. NO significantly impacts vasomotor tone to enhance conductance of the newly recruited collateral arteries, and this effect is augmented by exercise training prior to arterial occlusion. NO-mediated increases in vascular conductance allows for greater collateral dependent blood flow to the tissue distal to occlusion. NO production is also critical to the efficacy of therapeutic arteriogenesis achieved by delivery of exogenous angiogenic growth factors (VEGF, FGF-2) or by exercise training. The critical role of NO in therapeutic arteriogenesis is independent of NO-mediated changes in vascular conductance and implies a central role in arteriogenic signaling events. Maintenance, or improvement, of NO production and signaling, such as with regular exercise, may improve endothelial cell function and thus may help preserve the arteriogenic potential of preexisting collateral networks.

Relation of bone mineral density and content to mineral content and density of the fat-free mass.

Differences in the mineral fraction of the fat-free mass (M(FFM)) and in the density of the FFM (D(FFM)) are often inferred from measures of bone mineral content (BMC) or bone mineral density (BMD). We studied the relation of BMC and BMD to the M(FFM) and D(FFM) in a heterogeneous sample of 216 young men (n = 115) and women (n = 101), which included whites (n = 155) and blacks (n = 61) and collegiate athletes ( n = 132) and nonathletes (n = 84). Whole body BMC and BMD were determined by dual-energy X-ray absorptiometry (DXA; Hologic QDR-1000W, enhanced whole body analysis software, version 5.71). FFM was estimated using a four-component model from measures of body density by hydrostatic weighing, body water by deuterium dilution, and bone mineral by DXA. There was no significant relation of BMD to M(FFM) (r = 0.01) or D(FFM) (r = -0.06) or of BMC to M(FFM) (r = -0.11) and a significant, weak negative relation of BMC to D(FFM) (r = -0.14, P = 0.04) in all subjects. Significant low to moderate relationships of BMD or BMC to M(FFM) or D(FFM) were found within some gender-race-athletic status subgroups or when the effects of gender, race, and athletic status were held constant using multiple regression, but BMD and BMC explained only 10-17% of the variance in M(FFM) and 0-2% of the variance in D(FFM) in addition to that explained by the demographic variables. We conclude that there is not a significant positive relation of BMD and BMC to M(FFM) or D(FFM) in young adults and that BMC and BMD should not be used to infer differences in M(FFM) or D(FFM).

Biarticular and monoarticular muscle activation and injury in human quadriceps muscle.

We hypothesized that activation of the quadriceps femoris muscle group during eccentric exercise is related to the increase in magnitude of several markers of muscle injury that developed during the next week. Fourteen male subjects performed six to eight sets of five to ten repetitions of single-leg eccentric-only seated knee extension exercise. Magnetic resonance (MR) images were collected before and immediately after exercise and on days 2-4 and 6 after eccentric exercise. Changes in maximal voluntary contraction (MVC), perceived soreness, muscle volume and muscle transverse relaxation of water protons (T2) were determined for the quadriceps femoris muscle group each day. Changes in muscle volume and T2 were determined every day for each muscle [vastus lateralis (VL), vastus medialis (VM), vastus intermedius (VI), rectus femoris (RF)] of the quadriceps femoris group. Post-exercise T2 was greater than pre-exercise T2 (P < 0.05) for all muscles. The acute deltaT2 (Post-Pre) was similar (P>0.05) among VL, VM, VI, and RF [5.5 (0.3) ms], suggesting that the four muscles were equally activated during eccentric exercise. In the week after eccentric exercise, subjects experienced delayed-onset muscle soreness (DOMS) and all muscles demonstrated a delayed increase in T2 above pre-exercise values (P < 0.05), suggesting that muscle injury had occurred. For the quadriceps femoris muscle group, there was no correlation between acute deltaT2 and delayed (peak T2 during days 2, 3, 4, 6 minus pre-exercise T2) deltaT2 (r=0.04, P>0.05). Similar results were obtained when VL, VM, VI and RF were examined separately. Of the four muscles in quadriceps femoris, the biarticular RF experienced greater muscle injury [delayed deltaT2= 15.2 (2.0) ms] compared to the three monoarticular vasti muscles [delayed deltaT2 = 7.7 (1.3) ms; P< 0.05]. We propose that the disproportionate muscle injury to RF resulted from an ineffective transfer of torque from the knee to hip joint during seated eccentric knee extension exercise, thus causing RF to dissipate greater energy than normal. We conclude that in humans, muscle activation is not a unique determinant of muscle injury.

Contraction increases the T(2) of muscle in fresh water but not in marine invertebrates.

Previous studies suggest that the activity-induced increase in (1)H-NMR transverse relaxation time (T(2)) observed in mammalian skeletal muscles is related to an osmotic effect of intracellular metabolite accumulation. This hypothesis was tested by comparing T(2) (measured by (1)H-NMR imaging at 4.7 T) and metabolite changes (measured by (31)P-NMR spectroscopy) after stimulation in the muscles of a freshwater (crayfish, Orconectes virilis) vs two osmoconforming marine invertebrates (lobster, Homarus americanus; scallop, Argopecten concentricus). Intracellular pH significantly decreased after stimulation in the lobster tail muscle, but not in the crayfish tail or scallop phasic adductor muscles. The decrease in phosphoarginine-to-ATP ratio after stimulation was similar in the three muscles. Muscle T(2) increased from 37 to 43 ms (p < 0.02, n = 7) after stimulation in crayfish, but was unchanged in lobster muscle (32 ms, n = 7), and significantly decreased (from 40 to 36 ms, p < 0.02, n = 11) in scallop muscle. The observation that T(2) does not increase after stimulation in muscles of marine invertebrates with high natural osmolarity is consistent with the hypothesis that the T(2) increase in mammalian muscle is related to osmotically driven shifts of fluid between subcellular compartments.

Muscularity and the density of the fat-free mass in athletes.

The purpose of this study was to use estimates of body composition from a four-component model to determine whether the density of the fat-free mass (D(FFM)) is affected by muscularity or musculoskeletal development in a heterogenous group of athletes and nonathletes. Measures of body density by hydrostatic weighing, body water by deuterium dilution, bone mineral by whole body dual-energy X-ray absorptiometry (DXA), total body skeletal muscle estimated from DXA, and musculoskeletal development as measured by the mesomorphy rating from the Heath-Carter anthropometric somatotype were obtained in 111 collegiate athletes (67 men and 44 women) and 61 nonathletes (24 men and 37 women). In the entire group, D(FFM) varied from 1.075 to 1.127 g/cm3 and was strongly related to the water and protein fractions of the fat-free mass (FFM; r = -0.96 and 0.89) and moderately related to the mineral fraction of the FFM (r = 0.65). Skeletal muscle (%FFM) varied from 40 to 68%, and mesomorphy varied from 1.6 to 9.6, but neither was significantly related to D(FFM) (r = 0.11 and -0.14) or to the difference between percent fat estimated from the four-component model and from densitometry (r = 0.09 and -0.16). We conclude that, in a heterogeneous group of young adult athletes and nonathletes, D(FFM) and the accuracy of estimates of body composition from body density using the Siri equation are not related to muscularity or musculoskeletal development. Athletes in selected sports may have systematic deviations in D(FFM) from the value of 1.1 g/cm3 assumed in the Siri equation, resulting in group mean errors in estimation of percent fat from densitometry of 2-5% body mass, but the cause of these deviations is complex and not simply a reflection of differences in muscularity or musculoskeletal development.

Effect of aerobic capacity on the T(2) increase in exercised skeletal muscle.

The increase in nuclear magnetic resonance transverse relaxation time (T(2)) of muscle water measured by magnetic resonance imaging after exercise has been correlated with work rate in human subjects. This study compared the T(2) increase in thigh muscles of trained (cycling VO(2 max) = 54.4 +/- 2.7 ml O(2). kg(-1). min(-1), mean +/- SE, n = 8, 4 female) vs. sedentary (31.7 +/- 0.9 ml O(2). kg(-1). min(-1), n = 8, 4 female) subjects after cycling exercise for 6 min at 50 and 90% of the subjects' individually determined VO(2 max). There was no significant difference between groups in the T(2) increase measured in quadriceps muscles within 3 min after the exercises, despite the fact that the absolute work rates were 60% higher in the trained group (253 +/- 15 vs. 159 +/- 21 W for the 90% exercise). In both groups, the increase in T(2) of vastus muscles was twofold greater after the 90% exercise than after the 50% exercise. The recovery of T(2) after the 90% exercise was significantly faster in vastus muscles of the trained compared with the sedentary group (mean recovery half-time 11.9 +/- 1.2 vs. 23.3 +/- 3.7 min). The results show that the increase in muscle T(2) varies with work rate relative to muscle maximum aerobic power, not with absolute work rate.

Fiber type and metabolic dependence of T2 increases in stimulated rat muscles.

This study examined the relationships between muscle fiber type, metabolism, and blood flow vs. the increase in skeletal muscle (1)H-NMR transverse relaxation time (T2) after stimulation. Triceps surae muscles of anesthetized rats were stimulated in situ at 1-10 Hz for 6 min, and T2 was calculated from (1)H-NMR images acquired at 4.7 T immediately after stimulation. At low-to-intermediate frequencies (1-5 Hz), the stimulation-induced T2 increase was greater in the superficial, fast-twitch white portion of the gastrocnemius muscle compared with the deeper, more aerobic muscles of the triceps surae group. Although whole triceps muscle area changed in parallel with T2 after stimulation when blood flow was intact, clamping of the femoral artery during stimulation prevented an increase in muscle area but not an increase in T2. Partial inhibition of lactic acid production with iodoacetate diminished intracellular acidification (measured by (31)P-NMR spectroscopy) during brief (1.5 min) stimulation but had no significant effect either on estimated osmolite accumulation or on muscle T2 after stimulation. Depletion of muscle phosphocreatine content by feeding rats beta-guanidinopropionate decreased both estimated osmolite accumulation and T2 after 1.5-min stimulation. The results are consistent with the hypothesis that the T2 increase in stimulated muscle is related to osmotically driven shifts of fluid into an intracellular compartment.

Quantification of plasma membrane ergosterol of Saccharomyces cerevisiae by direct-injection atmospheric pressure chemical ionization/tandem mass spectrometry.

A method for the quantification of ergosterol by atmospheric pressure chemical ionization (APcI) mass spectrometry with direct injection is described. Ergosterol and squalene were ionizable with methanol as the carrier solvent. Using positive-mode tandem mass spectrometry (MS/MS), ergosterol could be identified unambiguously without interference from structurally related compounds such as lanosterol, cholesterol, and squalene. Molecular ions of ergosterol, lanosterol, and cholesterol were detected as the [M + H - H(2)O](+) ion species, while squalene appeared as the [M + H](+) ion species. Upon fragmentation of the three sterols and squalene, the product ion at m/z 69 was present as one of the major fragments in all four compounds. This product ion was used for the quantification of ergosterol in multiple-reaction-monitoring acquisition mode. The relationship between signal intensity and ergosterol concentration was linear over the concentration range of 0.15 to 5 microg/ml, or 7. 56-252 pmol ergosterol per 20 microl injection. The plasma membrane ergosterol of the yeast Saccharomyces cerevisiae could be quantified reproducibly without the need for prior separation from other lipids or derivatization. Six repeated injections of ergosterol standards at concentrations of 0.95 and 4.25 microg/ml gave standard deviations of 0.031 and 0.084, respectively, and coefficients of variation of 3.33 and 1.98%, respectively. The coefficient of variation for the four independently extracted membrane ergosterol samples was 11.18%. The presence of other lipids in a crude lipid extract did not interfere with the ergosterol determination. Direct injection APcI with multiple reaction monitoring is aconvenient and sensitive method for ergosterol quantification requiring no prior fractionation.

Conservation and release of osmolytes by yeasts during hypo-osmotic stress.

In response to fluctuations in environmental osmolarity, yeast cells adjust their intracellular solute concentrations in order to maintain a constant turgor pressure and ensure continuation of cellular activity. In this study, the effect of hypo-osmotic stress on osmolyte content of osmotolerant yeasts Zygosaccharomyces rouxii and Pichia sorbitophila and the less tolerant Saccharomyes cerevisiae was investigated. All these yeasts released glycerol upon hypo-osmotic shock. However, Z. rouxii also released arabitol, whereas P. sorbitophila released erythritol in addition to arabitol and glycerol. Osmolyte release was very rapid and specific and was neither affected by reduced temperatures nor inhibited by the channel blocker gadolinium or the protonophore carbonyl cyanide m-chlorophenyl hydrazone. Extracellular osmolyte levels increased drastically suggesting that osmolytes were not metabolised but mainly released upon exposure to hypotonic conditions. The export process is well controlled, and the amount of osmolyte released was proportional to the shock intensity. Osmolyte release occurred with little cell lysis and thus the survival as well as the subsequent growth of yeast cells was largely unaffected after hypo-osmotic shock. The kinetics and patterns of osmolyte export suggest the involvement of channel proteins, but the molecular nature of this export pathway in yeasts, with exception of S. cerevisiae, remains to be established.