Phase transitions in thylakoid polar lipids of chilling-sensitive plants: a comparison of detection methods

The phase behavior of thylakoid polar lipids from plants sensitive to chilling injury was investigated by calorimetry, electron spin resonance spectroscopy of spin labels, and fluorescence intensity after labeling with trans-parinaric acid. The plants used were oleander (Nerium oleander), mung bean (Vigna radiata L. var Mungo), and tomato (Lycopersicon esculentum cv Grosse Lisse). For all plants the initiation temperature for the calorimetric exotherm was coincident (±1°C) with the transition determined by the increase in the temperature coefficient of spin label motion and fluorescence intensity of trans-parinaric acid. For oleander plants, grown at 45°C, the transition was at 7°C while for plants from the same clone, grown at 20°C, it was at −2°C. For mung bean and tomato the transition was between 9 and 12°C. The similarity in the transition detected by spin labeling and fluorescence intensity suggest that spin labels, like the fluorescent label trans-parinaric acid, preferentially partition into domains of ordered lipid. The coincidence of the temperature for initiation of the transition, determined by the three techniques, shows that each is a valid method of assessing a phase transition in membrane polar lipids.     

The Formation and Distribution of Ice within Forsythia Flower Buds

Differential thermal analysis detected two freezing events when dormant forsythia (Forsythia viridissima Lindl.) flower buds were cooled. The first occurred just below 0°C, and was coincident with the freezing of adjacent woody tissues. The second exotherm appeared as a spike between −10 and −25°C and was correlated with the lethal low temperature. Although this pattern of freezing was similar to that observed in other woody species, differences were noted. Both direct observations of frozen buds and examination of buds freeze-fixed at −5°C demonstrated that ice formed within the developing flowers at temperatures above the second exotherm and lethal temperature. Ice crystals had formed within the peduncle and in the lower portions of the developing flower. Ice also formed within the scales. In forsythia buds, the developing floral organ did not freeze as a unit as noted in other species. Instead the low temperature exotherm appeared to correspond to the lethal freezing of supercooled water within the anthers and portions of the pistil.     

Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes

Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 ± 1.7 years) and middle-aged (mean age = 56.3 ± 4.7 years) subjects after 1 hour of heat shock at 42°C. Genotype-specific HSR was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible and inducible forms of Hsp70 decreased 1.3-fold (P < 0.001) and 1.4-fold (P < 0.001), respectively, in the monocytes with age. In the young subjects, a positive association was found between HSPA1L(T2437C) polymorphism and HSR. CC carriers had a significantly lower induction than TT carriers in both monocytes (P = 0.015) and lymphocytes (P = 0.044). This polymorphism, which is present in the coding region of HSPA1L gene, can affect the chaperoning function of Hsp70. These data consolidate our other observations that the CC genotype is unfavorable for human longevity and provide a functional explanation in terms of variations in HSR.     

N-Substituted Benzyl Matrinic Acid Derivatives Inhibit Hepatitis C Virus (HCV) Replication through Down-Regulating Host Heat-Stress Cognate 70 (Hsc70) Expression

Heat-stress cognate 70 (Hsc70) is a host factor that helps hepatitis C virus (HCV) to complete its life cycle in infected hepatocytes. Using Hsc70 as a target for HCV inhibition, a series of novel N-substituted benzyl matrinic/sophoridinic acid derivatives was synthesized and evaluated for their anti-HCV activity in vitro. Among these analogues, compound 7c possessing N-p-methylbenzyl afforded an appealing ability to inhibit HCV replication with SI value over 53. Furthermore, it showed a good oral pharmacokinetic profile with area-under-curve (AUC) of 13.4 µM·h, and a considerably good safety in oral administration in mice (LD₅₀>1000 mg/kg). As 7c suppresses HCV replication via an action mode distinctly different from that of the marketed anti-HCV drugs, it has been selected as a new mechanism anti-HCV candidate for further investigation, with an advantage of no or decreased chance to induce drug-resistant mutations.     

Monomeric Banana Lectin at Acidic pH Overrules Conformational Stability of Its Native Dimeric Form

Banana lectin (BL) is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS) binding, size exclusion chromatography (SEC) and dynamic light scattering (DLS). During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml) at pH 2.0 while single peak (61.45 ml) at pH 7.4. The hydrodynamic radii (R _h) of native BL was 2.9 nm while at pH 2.0 two species were found with R _h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high C_m and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.     

Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence

The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32°C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30°C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32°C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14°C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32°C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.     

Influence of Water and Temperature Stress on the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination

The temperature dependence of the rate and magnitude of the reappearance of photosystem II (PSII) variable fluorescence following illumination has been used to determine plant temperature optima. The present study was designed to determine the effect of a plant's environmental history on the thermal dependency of the reappearance of PSII variable fluorescence. In addition, this study further evaluated the usefulness of this fluorescence technique in identifying plant temperature optima. Laboratory and greenhouse grown potato (Solanum tuberosum L. cv “Norgold M”) plants had a thermal kinetic window between 15 and 25°C. The minimum apparent K_m of NADH hydroxypyruvate reductase for NADH occurred at 20°C. This temperature was also the temperature providing maximal reappearance of variable fluorescence. Soybean (Glycine max [L.] Merrill cv “Wayne”) plants had a thermal kinetic window between 15 and 30°C with a minimum apparent K_m at 25°C. Maximal reappearance of variable fluorescence was seen between 20 and 30°C. To determine if increasing environmental temperatures increased the temperature optimum provided from the fluorescence response curves, potato and soybean leaves from irrigated and dryland field grown plants were evaluated. Although the absolute levels of PSII variable fluorescence declined with increasing thermal stress, the temperature optimum of the dryland plants did not increase with increased exposure to elevated temperatures. Because of variability in the daily period of high temperature stress in the field, studies were initiated with tobacco plants grown in controlled environment chambers. The reappearance of PSII variable fluorescence in tobacco (Nicotiana tabacum L. cv “Wisconsin 38”) leaves that had experienced continuous leaf temperatures of 35°C for 8 days had the same 20°C optima as leaves from plants grown at room temperature. The results of this study suggest that the temperature optimum for the reappearance of variable fluorescence following illumination is not altered by the plant's previous exposure to variable environmental temperatures. These findings support the usefulness of this procedure for the rapid identification of a plant's temperature optimum.     

Rapid increase in deep supercooling of xylem parenchyma

Malus pumila Mill. twigs were collected from September through December and stored at 5°C until the low temperature exotherms of the xylem were determined by differential thermal analysis. During the differential thermal analysis, cooling was interrupted, and temperatures of 5 to −18°C were held for 0.4 to 10 hours before cooling to −50°C was resumed. Control twigs were cooled to −50°C without interruption. Holding the twigs at 1.3 to −5°C shifted the start of the low temperature exotherm from about −20 to −30°C. Slightly higher (2.6°C) and lower (−10°C) temperatures were occasionally effective. The shift began within 20 to 30 minutes and increased progressively to 150 minutes. The acclimation was reversibly inhibited by N₂ atmosphere.     

A plant reovirus hijacks the DNAJB12–Hsc70 chaperone complex to promote viral spread in its planthopper vector

Many viruses usurp the functions of endoplasmic reticulum (ER) for virus‐encoded membrane proteins proper functional folding or assembly to promote virus spread. Southern rice black‐streaked dwarf virus (SRBSDV), a plant reovirus, exploits virus‐containing tubules composed of nonstructural membrane protein P7‐1 to spread in its planthopper vector Sogatella furcifera. Here, we report that two factors of the ER‐associated degradation (ERAD) machinery, the ER chaperone DNAJB12 and its cytosolic co‐chaperone Hsc70, are activated by SRBSDV to facilitate ER‐to‐cytosol export of P7‐1 tubules in S. furcifera. Both P7‐1 of SRBSDV and Hsc70 directly bind to the J‐domain of DNAJB12. DNAJB12 overexpression induces ER retention of P7‐1, but Hsc70 overexpression promotes the transport of P7‐1 from the ER to the cytosol to initiate tubule assembly. Thus, P7‐1 is initially retained in the ER by interaction with DNAJB12 and then delivered to Hsc70. Furthermore, the inhibitors of the ATPase activity of Hsc70 reduce P7‐1 tubule assembly, suggesting that the proper folding and assembly of P7‐1 tubules is dependent on the ATPase activity of Hsc70. The DNAJB12–Hsc70 chaperone complex is recruited to P7‐1 tubules in virus‐infected midgut epithelial cells in S. furcifera. The knockdown of DNAJB12 or Hsc70 strongly inhibits P7‐1 tubule assembly in vivo, finally suppressing effective viral spread in S. furcifera. Taken together, our results indicate that the DNAJB12–Hsc70 chaperone complex in the ERAD machinery facilitates the ER‐to‐cytosol transport of P7‐1 for proper assembly of tubules, enabling viral spread in insect vectors in a manner dependent on ATPase activity of Hsc70.     

Effectiveness of a Hot Water Drench for the Control of Foliar Nematodes Aphelenchoides fragariae in Floriculture

Effectiveness of a hot water drench for the control of Aphelenchoides fragariae infesting hosta (Hosta sp.) and ferns (Matteuccia pensylvanica) was studied. Drenching with hot water at 70 °C and 90 °C in October reduced (P < 0.05) A. fragariae in the soil but not in the leaves relative to the control (25 °C) 300 days after treatment (DAT). Plants drenched with 90 °C water had lower numbers of nematode-infected leaves per plant than those treated with 25 °C and 70 °C water (P < 0.05). Hot water treatments had no adverse effect on the growth parameters of hosta. Boiling water (100 °C) applied once a month for 3 consecutive months (April, May, June) consistently reduced the number of infected leaves and the severity of infection relative to the control 150 DAT in hosta but not in ferns (P < 0.05). Boiling water (100 °C) caused a 67% reduction in A. fragariae population in hosta leaves, 50% in fern fronds, and 61% to 98% in the soil over the control 150 DAT. A boiling water drench had no effect on the fern growth but caused 49% and 22% reduction in the number and size of hosta leaves, respectively, over the control in 2002. We conclude that 90 °C water soil drench in the autumn or early spring could prove effective in managing foliar nematodes on hosta in nurseries and landscapes.     

Endosperm and pericarp involvement in the supercooling of imbibed lettuce seeds

Fully hydrated lettuce (Lactuca sativa L.) seeds showed dual freezing exotherms (−9 and −18°C), even after 10 hours imbibition. Only the −9°C exotherm was observed in seeds imbibed for 20 hours, but without external nucleation, all water in the embryo supercooled. Results indicate that the endosperm acts as a barrier to ice propagation. Other experiments suggest that the pericarp may also protect the embryo under certain freezing conditions.     

Variation among Species in the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination

The relationship between the thermal dependence of the reappearance of chlorophyll variable fluorescence following illumination and temperature dependence of the apparent Michaelis constant (K_m) of NADH hydroxypyruvate reductase for NADH was investigated in cool and warm season plant species. Brancker SF-20 and SF-30 fluorometers were used to evaluate induced fluorescence transients from detached leaves of wheat (Triticum aestivum L. cv TAM-101), cotton (Gossypium hirsutum L. cv Paymaster 145), tomato (Lycopersicon esculentum cv Del Oro), bell pepper (Capsicum annuum L. cv California Wonder), and petunia (Petunia hybrida cv. Red Sail). Following an illumination period at 25°C, the reappearance of variable fluorescence during a dark incubation was determined at 5°C intervals from 15°C to 45°C. Variable fluorescence recovery was normally distributed with the maximum recovery observed at 20°C in wheat, 30°C in cotton, 20°C to 25°C in tomato, 30 to 35°C in bell pepper and 25°C in petunia. Comparison of the thermal response of fluorescence recovery with the temperature sensitivity of the apparent K_m of hydroxypyruvate reductase for NADH showed that the range of temperatures providing fluorescence recovery corresponded with those temperatures providing the minimum apparent K_m values (viz. the thermal kinetic window).     

Defective Chylomicron Synthesis as a Cause of Delayed Particle Clearance in Diabetes?

Chylomicron metabolism is abnormal in diabetes and the chylomicron particle may play a very important role in atherosclerosis. The aim of this study was to examine the effect of diabetes on the metabolism of chylomicrons in cholesterol-fed alloxan diabetic and nondiabetic rabbits. Five diabetic rabbits and 5 control rabbits were given [¹⁴C]linoleic acid and [³H]cholesterol by gavage. Lymph was collected following cannulation of the lymph duct and radiolabelled chylomicrons were isolated by ultracentrifugation. The chylomicrons from each animal were injected into paired control and diabetic recipients. Lymph apolipoprotein (apo) B48, apo B100, and apo E were measured using sodium dodecyl sulfate–polyacrylamide gradient gel electrophoresis. Mean blood sugar of the diabetic donors and diabetic recipients were 19.7 ± 2.3 and 17.2 ± 3.2 mmol/L. Diabetic rabbits had significantly raised plasma triglyceride (10.8 ± 13.9 versus 0.8 ± 0.5 mmol/L, P < 0.02). There was a large increase in apo B48 in lymph chylomicrons in the diabetic donor animals (0.19 ± 0.10 versus 0.04 ± 0.02 mg/h, P < 0.01) and apo B100 (0.22 ± 0.15 versus 0.07 ± 0.07 mg/h, P < 0.05) and a reduction in apo E on the lymph chylomicron particle (0.27 ± 0.01 versus 0.62 ± 0.07 mg/mg apo B, P < 0.001). Diabetic recipients cleared both control and diabetic chylomicron triglyceride significantly more slowly than control recipients (P < 0.05). Clearance of control chylomicron cholesterol was delayed when injected into diabetic recipients compared to when these chylomicrons were injected into control recipients (P < 0.005). Clearance of diabetic chylomicron cholesterol was significantly slower when injected into control animals compared to control chylomicron injected into control animals (P < 0.02). In this animal model of atherosclerosis, we have demonstrated that diabetes leads to the production of an increased number of lipid and apo E–deficient chylomicron particles. Chylomicron particles from the control animals were cleared more slowly by the diabetic recipient (both triglyceride and cholesterol). The chylomicron particles obtained from the diabetic animals were cleared even more slowly when injected into the diabetic recipient. Although there was an initial delay in clearance of chylomicron triglyceride from the diabetic particle when injected into the control animals, the clearance over the first 15 minutes was not significantly different when compared to the control chylomicron injected into the control animal. On the other hand, the cholesterol clearance was significantly delayed. Thus, diabetes resulted in the production of an increased number of lipid- and apo E–deficient chylomicron particles. These alterations account, in part, for the delay in clearance of these particles.     

Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu,Labeo rohita,with special reference to molecular characterization of Grp78

Heat shock protein (Hsp) genes are stress-related genes that activate the host immune system during infection. Hsp genes expression in fish, studied during bacterial infections, is mostly confined to Hsp70 and Hsp90. The present study is an expression analysis of seven Hsp genes: Apg2, Hsp90, Hsp70, glucose-regulated protein 78 (Grp78), heat shock cognate 70 (Hsc70), Grp75, and Hsp30 during Aeromonas hydrophila infection in rohu, Labeo rohita. Forty-eight rohu juveniles were challenged with 3 × 10⁷ cfu bacteria per 20 g of body weight intraperitoneally. The expression of these genes was studied in infected liver, anterior kidney, and spleen tissues of rohu at different time periods: 0, 1, 3, 6, 12, 24, 48, 72 h, 7, and 15 days post-infection by qPCR. The Hsp gene modulation was greater in liver as compared to spleen and kidney tissues. Induced expression of Apg2, Hsp90, Grp78, Grp75, and Hsc70 was noticed during peak periods (3 to 24 h post-challenge) of bacterial infectivity. Hsp70 was found to be down-regulated during the process of infection. In contrast to the other six genes, Hsp30 showed a variable expression pattern in all three tissues. Grp78 was found to be the most highly (1,587-fold) expressed gene in liver at 12 h post-challenge. Further, molecular characterization of Grp78 revealed it to be a highly conserved Hsp gene, essential not only during infection but also during early developmental stages of rohu, and its expression was noticed in all organs studied except in gill tissues, which indicated its potential immune regulatory role during infection process.     

Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.     

Studies on the effects of subzero temperatures on the viability of spores of Aspergillus flavus. I. The effect of rate of warming

1. The survival of spores of Aspergillus flavus suspended in distilled water and cooled rapidly to –70 to –75°C. was found to depend primarily on the rate of subsequent warming of the frozen suspension. Only 7 per cent of the spores germinated following slow warming at 0.9°C. per minute, whereas about 75 per cent germinated following rapid warming at 700°C. per minute. 2. Viability was dependent on the rate at which the suspensions warmed from –70 to 0°C. (subzero warming), but was not dependent on the rate of thawing of the frozen water in which the spores were suspended. 3. The logarithm of the percentage of germination appeared to be a linear function of the logarithm of the rate of subzero warming when spores were warmed at rates ranging from 0.12 to 1000°C. per minute. 4. The lethal effects of slow warming from –70 to 0°C. were more pronounced between about –20 and 0°C. than between –70 and –20°C. In the former range of temperatures, the percentage of germination decreased sharply as slow warming progressed towards 0°C. 5. Slow warming from –70 to 0°C. was more harmful to the spores than was a 1 or 2 hour exposure to constant temperatures between –70 and 0°C. 6. Slow warming was found to be more harmful than rapid warming when spores were suspended in horse serum, 0.16 molal sodium chloride, or 0.29 molal sucrose as well as in distilled water.     

Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7°C and −3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20°C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7°C or −3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.

A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2°C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29°C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7°C (or −3°C) its completion. Because of the present results, however, this interpretation needs to be modified.

Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.

     

Comparison of the Thermostability Properties of Three Acid Phosphatases from Molds: Aspergillus fumigatus Phytase, A. niger Phytase, and A. niger pH 2.5 Acid Phosphatase

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70°C. After heat denaturation at temperatures up to 90°C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of ≥80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80°C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.     

A comparison of two commercially available ELISA methods for the quantification of human plasma heat shock protein 70 during rest and exercise stress

This study compared resting and exercise heat/hypoxic stress-induced levels of plasma extracellular heat shock protein 70 (eHSP70) in humans using two commercially available enzyme-linked immunosorbent assay (ELIS)A kits. EDTA plasma samples were collected from 21 males during two separate investigations. Participants in part A completed a 60-min treadmill run in the heat (HOT70; 33.0 ± 0.1 °C, 28.7 ± 0.8 %, n = 6) at 70 % V̇O_2max. Participants in part B completed 60 min of cycling exercise at 50 % V̇O_2max in either hot (HOT50; 40.5 °C, 25.4 relative humidity (RH)%, n = 7) or hypoxic (HYP50; fraction of inspired oxygen (F_IO₂) = 0.14, 21 °C, 35 % RH, n = 8) conditions. Samples were collected prior to and immediately upon termination of exercise and analysed for eHSP70 using EKS-715 high-sensitivity HSP70 ELISA and new ENZ-KIT-101 Amp’d™ HSP70 high-sensitivity ELISA. ENZ-KIT was superior in detecting resting eHSP70 (1.54 ± 3.27 ng·mL⁻¹; range 0.08 to 14.01 ng·mL⁻¹), with concentrations obtained from 100 % of samples compared to 19 % with EKS-715 assay. The ENZ-KIT requires optimisation prior to running samples in order to ensure participants fall within the standard curve, a step not required with EKS-715. Using ENZ-KIT, a 1:4 dilution allowed for quantification of resting HSP70 in 26/32 samples, with a 1:8 (n = 3) and 1:16 (n = 3) dilution required to determine the remaining samples. After exercise, eHSP70 was detected in 6/21 and 21/21 samples using EKS-715 and ENZ-KIT, respectively. eHSP70 was increased from rest after HOT70 (p < 0.05), but not HOT50 (p > 0.05) or HYP50 (p > 0.05) when analysed using ENZ-KIT. It is recommended that future studies requiring the precise determination of resting plasma eHSP70 use the ENZ-KIT (i.e. HSP70 Amp’d® ELISA) instead of the EKS-715 assay, despite additional assay development time and cost required.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.