Effect of temperature on the in vitro assembly of bacterial flagella

The temperature dependence for the rate of reconstitution or polymerization (k₊) at neutral pH of the protein, flagellin, to flagella was measured using Ostwald-type viscometers. Similarly, the kinetics for the reverse process, the thermally-induced depolymerization of flagella filaments to the flagellin monomer (k_?) was measured. The temperature at which k_? equals zero was used to define the thermal dissociation temperature or melting point of flagella filaments. The remarkable similarity of melting points obtained (36.8 ± 0.2 deg. C) for flagella isolated from three Salmonella strains (SJ670, SJ25 and SJ30 bearing H-antigen types i, 1.2 and e, n, x, respectively) suggests that the structural stability of these different protein filaments is also similar.On increasing the temperature between 12 and 28°C, k₊ increased smoothly and had a Q₁₀ of 1.8. Above 28.0, k₊ decreased rapidly and fell to zero at a temperature near 37°C, its precise value varying with the bacterial strain. This result supports the prior hypothesis (Gerber &; Noguchi, 1967) that on heating, a reversible co-operative transconformation occurs between different states of the protein; in one state, flagellin (M) can polymerize to flagella, whereas its conformational isomer(s) may do so with difficulty or not at all.For strains SJ25 and SJ30 the rates of polymerization and depolymerization both fall to zero near 37°C. Therefore, mixtures of monomer and flagella fragments (short polymers or “seeds”), in all ratios, appear to be in equilibrium at temperatures near this critical temperature, and neither polymerization of flagellin to flagella nor melting of polymers is apparent.Measurements made on flagella from strain SJ670 showed that k₊ and k_? approached zero at 45 and 37°C, respectively. Within this temperature range the conc entration of monomer in equilibrium with filaments was determined. By a null -point type experiment, solutions of monomer and seed were mixed to find the ratio that showed neither increases (polymerization) nor decreases (depolyme rization) in viscosity with time. An unexpected finding was that the temperature defines a critical monomer concentration, which exists in equilibrium with any concentration of filaments (and not the ratio of monomer-to-filament concentrations). Thus, the polymerization of fiagellin to flagella corresponds to a phase change akin to either crystallization or condensation.Application, of the Clapeyron-Clausius equation to the results obtained yields a heat of condensation of 70 kcal/mol of monomeric protein. The enthalpy change associated with M ? M_i is estimated as 110 kcal/mol of protein. Since the heat content of these various forms of flagella protein lies in the order M_i > F > M, by difference we estimate the enthalpy change for the conversion of monomers to polymers to be 40 kcal/mol of monomer.     

Reactive group-embedded affinity labeling reagent for efficient intracellular protein labeling

Affinity labeling of a target protein is a powerful method for chemical biology studies. However, it is still difficult to label intracellular proteins efficiently in living cells. We propose the novel design strategy of a reactive group-embedded affinity labeling reagent for efficient protein labeling. With FKBP12 as the model target protein, the ligand binding pocket-oriented labeling reagent could label intracellular protein, whereas protein surface-oriented reagent was ineffective for labeling in living cells, partially because of the intracellular protein fluctuation under the macromolecular crowding effects. These results provide new insight for efficient intracellular protein labeling.     

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) TNF decoy receptor 3 (TNFRSF6B)

Decoy receptor 3 (DcR3), a member of TNF receptor superfamily, is a soluble receptor without death domain and cytoplasmic domain, and secreted by cells and binds with FasL, LIGHT and TL1A. The principal function of DcR3 is the inhibition of apoptosis by the binding cytotoxic ligands. Expression of DcR3 has been reported in a wide array of normal human tissues as well as tumors and tumor cell lines. Recently, DcR3 was reported to modulate a variety of immune responses in mammals. TNFR or DcR3 has been identified in some teleost fishes. However, DcR3 is not reported in cartilaginous fish which is the lowest vertebrate possessing the adaptive immune system. Here we identified DcR3 cDNA in shark (Trsc-DcR3) from an SSH library prepared from peripheral white blood cells stimulated with PMA. Four cysteine-rich domains (CRDs) in common with TNF receptor family members are present in the Trsc-DcR3 sequence. The deduced amino acid sequence of Trsc-DcR3 showed highest identity with the chicken (50.4%), followed by human (46.8%) and rainbow trout (36.5%) DcR3. In a phylogenetic tree of known TNFRSF sequences, the Trsc-DcR3 grouped with the chicken and human DcR3. Trsc-DcR3 mRNA was detected strongly in the gill, moderately in the brain, and weakly in the kidney, thymus and leydig. These data strongly suggest that the gene encoding Trsc-DcR3 in banded dogfish is a homolog of the human gene. mRNA expression of Trsc-DcR3 in the thymus and leydig suggests that DcR3 may act as a modulator in the immune system even at the phylogenetic level of cartilaginous fish.     

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.     

Effects of encouraged water drinking on thermoregulatory responses after 20 days of head-down bed rest in humans

We tested the hypothesis that encouraged water drinking according to urine output for 20 days could ameliorate impaired thermoregulatory function under microgravity conditions. Twelve healthy men, aged 24 ± 1.5 years (mean ± SE), underwent −6° head-down bed rest (HDBR) for 20 days. During bed rest, subjects were encouraged to drink the same amount of water as the 24-h urine output volume of the previous day. A heat exposure test consisting of water immersion up to the knees at 42°C for 45 min after a 10 min rest (baseline) in the sitting position was performed 2 days before the 20-day HDBR (PRE), and 2 days after the 20-day HDBR (POST). Core temperature (tympanic), skin temperature, skin blood flow and sweat rate were recorded continuously. We found that the −6° HDBR did not increase the threshold temperature for onset of sweating under the encouraged water drinking regime. We conclude that encouraged water drinking could prevent impaired thermoregulatory responses after HDBR.     

Abnormalities in aggression and anxiety in transgenic mice overexpressing activin E

To study the function of activin E, a TGF-β superfamily member, in the regulation of affective behavior, we investigated the behavior of transgenic mice overexpressing activin E (TgActβE mice). Male TgActβE mice showed aggressive behavior in resident-intruder tests. In elevated plus-maze tests, the percentage of open arm entries was significantly increased in female TgActβE mice compared with that in wild-type mice. Furthermore, female TgActβE mice stayed in the central area for a significantly longer time than wild-type mice in open field tests. These results indicated that TgActβE mice had less anxiety-like behavior. The number of restraint-stress-evoked c-Fos-positive cells in the hypothalamic paraventricular nucleus in TgActβE mice was significantly decreased compared with that in wild-type mice. This suggests that synthesis of corticotrophin-releasing hormone induced by stress was decreased in TgActβE mice. Taking these results together, activin E may act as a regulator of the hypothalamic-pituitary-adrenal axis.     

The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication

The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.     

Contribution of central versus sweat gland mechanisms to the seasonal change of sweating function in young sedentary males and females

In summer and winter, young, sedentary male (N = 5) and female (N = 7) subjects were exposed to heat in a climate chamber in which ambient temperature (Ta) was raised continuously from 30 to 42°C at a rate of 0.1°C min⁻¹ at a relative humidity of 40%. Sweat rates (SR) were measured continuously on forearm, chest and forehead together with tympanic temperature (Tty), mean skin temperature ( [`T] s ) \left( {\overline {\hbox{T}} {\hbox{s}}} \right) and mean body temperature ( [`T] b ) \left( {\overline {\hbox{T}} {\hbox{b}}} \right) . The rate of sweat expulsions (Fsw) was obtained as an indicator of central sudomotor activity. Tty and ( [`T] b ) \left( {\overline {\hbox{T}} {\hbox{b}}} \right) were significantly lower during summer compared with winter in males; SR was not significantly different between summer and winter in males, but was significantly higher during summer in females; SR during winter was higher in males compared with females. The regression line relating Fsw to ( [`T] b ) \left( {\overline {\hbox{T}} {\hbox{b}}} \right) shifted significantly from winter to summer in males and females, but the magnitude of the shift was not significantly different between the two subject groups. The regression line relating SR to Fsw was steepened significantly from winter to summer in males and females, and the change in the slope was significantly greater in females than in males. Females showed a lower slope in winter and a similar slope in summer compared to males. It was concluded that sweating function was improved during summer mediated by central sudomotor and sweat gland mechanisms in males and females, and, although the change of sweat gland function from winter to summer was greater in females as compared with males, the level of increased sweat gland function during summer was similar between the two subject groups.