Positioning of the Alzheimer Aβ(1–40) peptide in SDS micelles using NMR and paramagnetic probes

NMR spectroscopy combined with paramagnetic relaxation agents was used to study the positioning of the 40-residue Alzheimer Amyloid β-peptide Aβ(1–40) in SDS micelles. 5-Doxyl stearic acid incorporated into the micelle or Mn²⁺ ions in the aqueous solvent were used to determine the position of the peptide relative to the micelle geometry. In SDS solvent, the two α-helices induced in Aβ(1–40), comprising residues 15–24, and 29–35, respectively, are surrounded by flexible unstructured regions. NMR signals from these unstructured regions are strongly attenuated in the presence of Mn²⁺ showing that these regions are positioned mostly outside the micelle. The central helix (residues 15–24) is significantly affected by 5-doxyl stearic acid however somewhat less for residues 16, 20, 22 and 23. This α-helix therefore resides in the SDS headgroup region with the face with residues 16, 20, 22 and 23 directed away from the hydrophobic interior of the micelle. The C-terminal helix is protected both from 5-doxyl stearic acid and Mn²⁺, and should be buried in the hydrophobic interior of the micelle. The SDS micelles were characterized by diffusion and ¹⁵N-relaxation measurements. Comparison of experimentally determined translational diffusion coefficients for SDS and Aβ(1–40) show that the size of SDS micelle is not significantly changed by interaction with Aβ(1–40). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Temperature-dependent sensitivity enhancement of solid-state NMR spectra of α-synuclein fibrils

The protein α-synuclein (AS) is the primary fibrillar component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Wild-type human AS and the three mutant forms linked to Parkinson’s disease (A53T, A30P, and E46K) all form fibrils through a nucleation-dependent pathway; however, the biophysical details of these fibrillation events are not yet well understood. Atomic-level structural insight is required in order to elucidate the potential role of AS fibrils in Parkinson’s disease. Here we show that low temperature acquisition of magic-angle spinning NMR spectra of wild type AS fibrils-greatly enhances spectral sensitivity, enabling the detection of a substantially larger number of spin systems. At 0 ± 3°C sample temperature, cross polarization (CP) experiments yield weak signals. Lower temperature spectra (−40 ± 3°C) demonstrated several times greater signal intensity, an effect further amplified in 3D ¹⁵N–¹³C–¹³C experiments, which are required to perform backbone assignments on this sample. Thus 3D experiments enabled assignments of most amino acids in the rigid part of the fibril (approximately residues 64 to 94), as well as tentative site-specific assignments for T22, V26, A27, Y39, G41, S42, H50, V52, A53, T54, V55, V63, A107, I112, and S129. Most of these signals were not observed in 2D or 3D spectra at 0 ± 3°C. Spectra acquired at low temperatures therefore permitted more complete chemical shift assignments. Observation of the majority of residues in AS fibrils represents an important step towards solving the 3D structure.     

Cell-substrate separation: effect of applied force and temperature

We measure the change in cell-substrate separation in response to an upward force by combining two relatively new techniques, Electric Cell-substrate Impedance Sensing (ECIS) to measure average cell-substrate separation, and collagen-coated magnetic beads to apply force to the top (dorsal) surface of cells. The collagen-coated ferric oxide beads attach to integrin receptors in the dorsal surfaces of osteoblastlike ROS 17/2.8 cells. Magnetic force is controlled by the position and the number of permanent magnets, applying an average 320 or 560 pN per cell. Comparing model calculations with experimental impedance data, the junctional resistivity of the cell layer and the average distance between the lower (ventral) cell surface and substrate can be determined. The ECIS analysis shows that these forces produce an increase in the distance between the ventral cell surface and the substrate that is in the range of 10 to 25%. At temperatures of 4°, 22° and 37 °C, the measured cell surface-substrate distances without magnetic beads are 84 ± 4, 45 ± 2 and 38 ± 2 nm. The force-induced changes at 22° are 11 ± 3 and 21 ± 3 nm for 320 and 560 pN, and at 37° they are 5 ± 2 and 9 ± 2 nm. The resulting cell-substrate spring constants at 22° and 37° are thus about 28 and 63 pN nm^–1 (dyne cm^–1). Using a reasonable range for the number for individual integrin-ligand adhesion bonds gives a range for the spring constant of the individual adhesion bond of from about 10^–3 to 10^–1 pN nm^–1. These data also provide evidence that the number of adhesion bonds per cell increases with temperature. Received: 20 June 1997 / Accepted: 24 August 1997     

Conformation of thymosin β9 in water/fluoroalcohol solution determined by NMR spectroscopy

The conformation of thymosin β₉ in solution of 40% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol-d₂ in water has been investigated by two-dimensional ¹H-nmr spectroscopy. Under this condition thymosin β₉ adopts an ordered structure. The determination of the conformation of the peptide was based on a set of 304 approximate interproton distance constraints derived from nuclear Overhauser enhancement measurements. The conformation of thymosin β₉ includes two helical regions from residues 4 to 27 and 32 to 41. The two helices are separated by a poorly defined loop region between amino acids 28 and 31; the N-terminus of thymosin B₉ shows random-coil structure only. © 1997 John Wiley & Sons, Inc. Biopoly 41: 623–634, 1997     

Zinc and copper intake and sources in healthy adults living in Bangkok and surrounding districts

The levels of intake and food sources of zinc and copper in 231 20–80 yr old healthy adults living in Bangkok and surrounding districts were determined. Dietary data from 3-d food records collected and validated at Research Center, Ramathibodi Hospital were analyzed. Levels of zinc and copper intake were compared between sexes and age groups (20–39, 40–59, and 60–80 yr). Mean daily zinc and copper intakes (±SEM) in men were 6.3 ±0.2 and 1.9 ±0.1 mg, respectively. Mean daily zinc and copper intakes (±SEM) estimated in women (5.5 ±0.2 and 1.6 +-0.1 mg) were significantly lower. Higher zinc and copper intakes were found in the younger (20-39 yr) age group of both sexes. This could be explained by higher density and percentage from animal source of both nutrients. Consumption of various types of meat, fish, egg, and milk accounted for 42 and 22%, and rice, the staple food of Thai people for 9 and 23% of total dietary zinc and copper, respectively.     

A contribution to the world selenium map

Atomic absorption spectrophotometric method was used to determine the serum selenium levels of 86 healthy individuals. Variations in age, sex, and geographically different urban regions of Yugoslavia were investigated. A group of 63 healthy children, ages 8–15 yr, were examined. Mean±standard deviation of the serum selenium concentration was 57±9 μg/L; age and geographic area had no effect on the Se status of children, but the difference between boys and girls was significant (P<0.05). A group of 23 men from Zagreb, ages 22–37 yr, were examined. The group was divided into three age subgroups and no difference was found among these groups. The mean Se concentration was 69±18 μg/L, and a statistically significant difference was found only between the group of adults and the group of children (P<0.05).     

Selenium levels in blood of Upper Silesian population

The selenium status and the relationship of whole-blood selenium and plasma homocysteine are reported for healthy human subjects living in Upper Silesia. A total of 1063 individuals (627 male and 436 female) examined for whole-blood selenium were subdivided into six groups according to age; the youngest included adolescents (n=143) aged 10–15 yr, and the oldest were centenarians (n=132). The mean Se content was relatively low (62.5±18.4 μg/L), and it tended to be higher in men (65.9±17.2 μg/L) than in women (57.5±18.9 μg/L). Selenium levels appeared to be age dependent, as the highest values were observed in young and middle-age adults (21–40 yr), whereas they were significantly lower in adolescents and in the elderly. In more than 40% of apparently healthy adults (aged 21–69 yr), the Se concentration was within the range 60–80 μg/L (i.e., below the lower limit of the nutritional adequacy range [80 μg/L]). A significant inverse correlation between whole-blood selenium and plasma total homocysteine was detected in a smaller population sample of middle-aged and elderly persons (n=204).     

The influence of snowfall,temperature and social relationships on sleeping clusters of Japanese monkeys during winter in Shiga Heights

We studied Japanese monkeys (Macaca fuscata) of the Shiga A₁ troop at their sleeping sites in Shiga Heights, Japan, for 41 nights during 3 winters. Monkeys chose their sleeping sites in Japanese cedars and in deciduous broad-leaved forests on non-snowing nights and in Japanese cedar forests on snowing nights. We counted 399 sleeping clusters in which 2 or more monkeys remained in physical contact through the night and 43 solitary sleeping monkeys, though monkeys did not maintain physical contact with others in the daytime. We found 397 clusters on tree branches and 2 clusters on rocks. The mean size of huddling clusters was 3.06±1.22 SD. The cluster size (3.17±1.26 SD) at lower ambient temperatures between −7 and −4°C was larger than that at higher temperatures between −2 and 4°C (cluster size 2.88±1.13 SD). Most clusters were composed of kin. Females kept close to related females in the daytime and huddled with them at night. The highest-ranking male mainly huddled with his kin and his familiar females. Other males kept farther apart from each other in the daytime, probably to avoid social conflicts. Through cold winter nights, however, such males reduced inter-individual distances and huddled with other males. Japanese monkeys appear to recognize three types of inter-individual distances: an intimate distance less than 1 m, a personal distance of 1–3 m and a social distance of 3–20 m; they change their inter-individual distances according to social and ecological circumstances.     

Flight potential of <Emphasis Type="Italic">Microplitis mediator</Emphasis>, a parasitoid of various lepidopteran pests

The efficacy of using natural enemies to control pests under field conditions largely depends on their mobility and, more specifically, on their capacity to quickly locate pest infestation. For many natural enemies, for example parasitoids, mobility is directly related to flight aptitude, which is determined by the capacity and inclination of a species to engage in flight. In this study, we determined the various factors that affected flight performance of Microplitis mediator (Haliday) (Hymenoptera: Braconidae), using a computer-monitored flight mill. No differences were found in flight performance (i.e., flight distance, duration, speed) between both sexes of M. mediator, and flight capacity increased up to an age of 5–7 days followed by a gradual decline afterwards. For one-day-old female parasitoids, mean (±SE) flight distance and duration were 6.23 ± 0.88 km and 85.15 ± 14.44 min, respectively, with a maximum flight distance of 18.0 km. For male parasitoids, mean flight distance and duration were 5.27 ± 0.51 km and 85.74 ± 7.63 min, respectively. Mating status did not affect flight performance of males, while flight distance of four-day-old ovipositing M. mediator females was much lower than that of un-mated females of the same age. Un-mated adults flew most actively at 22–24°C and inclination to fly gradually declined with decreasing temperature. Temperatures above 26°C considerably reduced flight activity of M. mediator. Wasps engaged in normal flight under a broad range of relative humidity (RH) conditions, with an optimum RH range identified as 75–90%. Our research shows that M. mediator is a highly active parasitoid, because both sexes show great inclination to fly under a range of environmental conditions and flight capacity at different ages. Our results can help explain parasitoid performance in the field and provide baseline information to help guide augmentative releases. Handling editor: Dirk Babendreier.     

The time-profile of the PBMC HSP70 response to in vitro heat shock appears temperature-dependent

Summary. Heat shock proteins (HSPs) are synthesised by cells subsequent to a stress exposure and are known to confer protection to the cell in response to a second challenge. HSP induction and decay are correlated to thermotolerance and may therefore be used as a biomarker of thermal history. The current study tested the temperature-dependent nature of the heat shock response and characterised its time profile of induction. Whole blood from 6 healthy males (Age: 26 ± (SD) 2 yrs; Body mass 74.2 ± 3.8 kgs; VO_2max: 49.1 ± 4.0 ml·kg⁻¹·min⁻¹) were isolated and exposed to in vitro heat shock (HS) at 37, 38, 39, 40, and 41 °C for a period of 90 min. After HS the temperature was returned to 37 °C and intracellular HSP70 was quantified from the leukocytes at 0, 2, 4, and 6 h after heat treatment. The concentration of HSP70 was not different between temperatures (P > 0.05), but the time-profile of HSP70 synthesis appeared temperature-dependent. At control (37 °C) and lower temperatures (38–39 °C) the mean HSP70 concentration increased up to 4 h post HS (P < 0.05) and then returned towards baseline values by 6 h post HS. With in vitro hyperthermic conditions (40–41 °C), the time-profile was characterised by a sharp rise in HSP70 levels immediately after treatment (P < 0.05 for 40 °C at 0 h), followed by a progressive decline over time. The results suggest a temperature-dependent time-profile of HSP70 synthesis. In addition, the temperature at which HSP70 is inducted might be lower than 37 °C.     

Inhibition of Botulinum Neurotoxin A Toxic Action In Vivo by Synthetic Synaptosome- and Blocking Antibody-Binding Regions

In previous studies, we showed that certain peptides of the H_N and H_C domains of the H-chain of BoNT/A bind to mouse brain synaptosomes (snps). There was either complete correspondence or overlap between peptides that bind snps and those that bind human or mouse blocking antibodies (Abs). An equimolar mixture of the overlapping peptides N5/N6/N7/N8 (residues 505–523/519–537/533–551/547–565) extended the survival time of the mice to 74 h (20%) relative to controls, which had a 50% survival time of 60 h. On the other hand, peptide N26 (residues 799–817) provided no protection (50% survival time, 58 h), but the overlapping peptide N25 (785–803) almost doubled the 50% survival time to 119 h. A mixture of the overlap N25/N26 provided an unexpected level of protection permitting 40% of the mice to survive a lethal BoNT/A dose. In the H_C domain, the overlap C23/C24 (1163–1181/1177–1195) provided no protection. Peptide C31 (1275–1296) also provided no significant protection. But an equimolar mixture of peptides C15/C16 (1051–1069/1065–1083) or peptides C18/C19/C20 (1093–1111/1107–1125/1121–1139) extended the 50% survival time by 41% (to 85 h) over controls (60 h) and was able to fully protect 20% of the mice which eventually recovered. Surprisingly, the mixture of the peptides C15/C16 and C18/C19/C20, which gave a 50% survival time of 75 h, was less protective than either peptides C15/C16 or peptides C18/C19/C20. The in vivo inhibitory activity of these peptides is discussed in relation to their location in the 3-dimensional structure of the toxin molecule and their membrane receptor binding.     

Effect of predator exposure on the performance and survival of red drum (<Emphasis Type="Italic">Sciaenops ocellatus</Emphasis>)

The influence of predator exposure on the survival of hatchery red drum, Sciaenops ocellatus was investigated under laboratory conditions. Several prey-capture (attack distance, mean attack velocity, capture time, and gape cycle duration) and anti-predator (reaction distance, response distance, mean velocity, and maximum velocity) performance variables were quantified using high-speed video for juvenile red drum (25–30 mm standard length, L _S ) reared with and without predators (pinfish, Lagodon rhomboides). Univariate contrasts of prey-capture events demonstrated that attack distance (mean ± s.e.) was significantly greater in red drum reared with (1.20 ± 0.16 mm) versus without (0.65 ± 0.09 mm) pinfish predators. During anti-predator events, red drum reared with predators had approximately 300% greater reaction distance and 20%–30% greater response distance, mean velocity and maximum velocity versus fish reared without predators; however, these differences were not statistically significant. Following video assessments, a series of mortality experiments were conducted using free-ranging pinfish predators. Mortality rates (Z) ranged from 0.047 to 0.060 (Z/hr/predator) and did not differ significantly among treatments.     

Role of brown bears (Ursus arctos) in the flow of marine nitrogen into a terrestrial ecosystem

We quantified the amount, spatial distribution, and importance of salmon (Oncorhynchus spp.)-derived nitrogen (N) by brown bears (Ursus arctos) on the Kenai Peninsula, Alaska. We tested and confirmed the hypothesis that the stable isotope signature (δ¹⁵N) of N in foliage of white spruce (Picea glauca) was inversely proportional to the distance from salmon-spawning streams (r=–0.99 and P<0.05 in two separate watersheds). Locations of radio-collared brown bears, relative to their distance from a stream, were highly correlated with δ¹⁵N depletion of foliage across the same gradient (r=–0.98 and –0.96 and P<0.05 in the same two separate watersheds). Mean rates of redistribution of salmon-derived N by adult female brown bears were 37.2±2.9 kg/year per bear (range 23.1–56.3), of which 96% (35.7±2.7 kg/year per bear) was excreted in urine, 3% (1.1±0.1 kg/year per bear) was excreted in feces, and <1% (0.3± 0.1 kg/year per bear) was retained in the body. On an area basis, salmon-N redistribution rates were as high as 5.1±0.7 mg/m² per year per bear within 500 m of the stream but dropped off greatly with increasing distance. We estimated that 15.5–17.8% of the total N in spruce foliage within 500 m of the stream was derived from salmon. Of that, bears had distributed 83–84%. Thus, brown bears can be an important vector of salmon-derived N into riparian ecosystems, but their effects are highly variable spatially and a function of bear density. Received: 11 February 1999 / Accepted: 7 July 1999     

Effects of elevated temperature on bacterial community structure and function in bioreactors treating a synthetic wastewater

The impact of elevated temperature on bacterial community structure and function during aerobic biological wastewater treatment was investigated. Continuous cultures, fed a complex growth medium containing gelatin and α-lactose as the principal carbon and energy sources, supported mixed bacterial consortia at temperatures ranging from 25–65°C. These temperature- and substrate-acclimated organisms were then used as inocula for batch growth experiments in which the kinetics of microbial growth and substrate utilization, efficiency of substrate removal, and mechanism of substrate removal were compared as functions of temperature. Bacterial community analysis by denaturing gradient gel electrophoresis (DGGE) revealed that distinct bacterial consortia were supported at each temperature. The efficiency of substrate removal declined at elevated temperatures. Maximum specific growth rates and the growth yield increased with temperature from 25–45°C, but then decreased with further elevations in temperature. Thus, maximum specific substrate utilization rates did not vary significantly over the 40°C temperature range (0.64 ± 0.04 mg COD mg⁻¹ dry cell mass h⁻¹). A comparison of the degradation of the protein and carbohydrate portions of the feed medium revealed a lag in α-lactose uptake at 55°C, whereas both components were utilized simultaneously at 25°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 140–145. Received 09 August 1999/ Accepted in revised form 12 November 1999     

The effect of time of day and exercise on platelet functions and platelet–neutrophil aggregates in healthy male subjects

Platelet activation state changes by exercise. The effect of exercise time on platelet activation state and formation of platelet–neutrophil aggregates are not known yet. In this study the effect of exercise and time of day were examined on platelet activity with platelet–neutrophil aggregates. Ten moderately active males aged 27± 1.63 (mean±S.D.) years completed sub-maximal (70% VO_2max) exercise trials for 30 min. Blood pressure (BP) was recorded. Venous blood samples were obtained at rest, immediately post-exercise and after 30 min of recovery. Whole blood was analysed for haematocrit (Hct), haemoglobin (Hb), platelet count (PC), mean platelet count (MPV) and platelet aggregation (PA). Platelet–neutrophil aggregates and beta-thromboglobulin (β-TG) levels were assayed. Platelet count showed significant increase after morning exercise ((236± 32)×10⁹ l⁻¹ versus (202± 34)×10⁹ l⁻¹ baseline, p < 0.05). Exercise resulted in significantly lower MPV after the evening exercise (9.16± 0.5 fl versus 9.65± 0.36 fl, p < 0.05). Platelet aggregation by adenosine diphosphate (ADP) decreased after morning exercise and the recovery aggregation levels were significantly different at two different times of the day (68± 20% a.m. versus 80± 12% p.m., p < 0.05). It was also showed that platelet–neutrophil aggregates increased significantly from baseline after both exercises. Exercise-induced platelet–neutrophil aggregates were higher in the evening (10.7± 1.3% p.m. versus 6.4± 1.8% a.m., p < 0.0001). It is therefore concluded that besides platelet–platelet aggregation, exercise can cause platelet– neutrophil aggregates. In addition, time of day has an effect on platelet activation related events. Circadian variations of physiological parameters may have an effect on thrombus formation by platelet activation. (Mol Cell Biochem xxx: 119–124, 2005)     

Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

Overstreetia olsoni n. sp. (Digenea: Zoogonidae) from the Capricorn silverside Atherinomorus capricornensis (Woodland) (Atherinidae) off Heron Island, southern Great Barrier Reef

A new species, Overstreetia olsoni, is described from Atherinomorus capricornensis off Heron Island on the Great Barrier Reef off Queensland, Australia. It differs from its only known congener in that the enlarged spines of the circum-oral rows are smaller ( ≤ 4.5 × 3.5 vs ≤ 21.5 × 10.5μm), the sucker-ratio is smaller (1: 0.97–1.01 vs 1:1.8) and the eggs are larger (40–45 × 22–28 vs 34–37 × 17–22μm).     

Specific characteristics of the strains isolated from a thermoacidophilic microbial community oxidizing antimony sulfide ore

Investigation of the phenotypic properties of three mixotrophic bacteria, strains Sb-K, Sb-F, and Sb-S, isolated from an aboriginal thermoacidophilic microbial community participating in biooxidation of ore with high antimony content (26%) and ore concentrates from the Olympiadinskoe deposit under semicontinuous cultivation conditions at 46 ± 1°C, revealed the differentiating characteristics of these strains. The isolated cultures grew lithotrophically through different numbers of transfers: strains Sb-F and Sb-K grew through seven and eight transfers, respectively, and strain Sb-S grew through two or three transfers. Strains Sb-K and Sb-S utilized a wide range of organic substrates for active organotrophic growth during nine or ten transfers, while strain Sb-F was less tolerant to organic compounds. Strain Sb-K grew on a medium with the ore and sulfide ore concentrates in the pH range of 1.0–3.0. Growth of strains Sb-F and Sb-S occurred in the pH ranges of 1.0–2.5 and 1.5–5.5 on media with Fe²⁺ and S⁰, respectively.. The optimal initial pH values of the media, corresponding to the maximum specific growth rates, were 1.6–1.7, 1.9, and 2.0–3.0 for strains Sb-K, Sb-F, and Sb-S, respectively. All three strains were able to grow within a broad temperature range, 20–65°C, with an optimum at 46°C (Sb-K), 40–46°C (Sb-F), and 48–50°C (Sb-S). According to the results of DNA-DNA hybridization and phylogenetic analysis, as well as their phenotypic characteristics, the isolates can be classified as novel strains of species of the genus Sulfobacillus. Strains Sb-K, Sb-F, and Sb-S, isolated as predominant cultures on the media with sulfide compounds, iron, or sulfur, respectively, were affiliated to the species S. thermotolerans, S. sibiricus, and S. thermosulfidooxidans.