Role of the loop L-4,L-5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L-4,L-5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L-4,L-5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L-4,L-5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein-bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.

Temperature-Induced Reversible First-Order Single Crystal to Single Crystal Phase Transition in Boc-gamma(4)(R)Val-Val-OH: Interplay of Enthalpy and Entropy

Crystals of Boc-gamma y(4)(R)Val-Val-OH undergo a reversible first-order single crystal to single crystal phase transition at T-c approximate to 205 K from the orthorhombic space group P22(1)2(1) (Z' = 1) to the monoclinic space group P2(1) (Z' = 2) with a hysteresis of similar to 2.1 K. The low-temperature monoclinic form is best described as a nonmerohedral twin with similar to 50% contributions from its two components. The thermal behavior of the dipeptide crystals was characterized by differential scanning calorimetry experiments. Visual changes in birefringence of the sample during heating and cooling cycles on a hot-stage microscope with polarized light supported the phase transition. Variable-temperature unit cell check measurements from 300 to 100 K showed discontinuity in the volume and cell parameters near the transition temperature, supporting the first-order behavior. A detailed comparison of the room-temperature orthorhombic form with the low-temperature (100 K) monoclinic form revealed that the strong hydrogen-bonding motif is retained in both crystal systems, whereas the non-covalent interactions involving side chains of the dipeptide differ significantly, leading to a small change in molecular conformation in the monoclinic form as well as a small reorientation of the molecules along the ac plane. A rigid-body thermal motion analysis (translation, libration, screw; correlation of translation and libration) was performed to study the crystal entropy. The reversible nature of the phase transition is probably the result of an interplay between enthalpy and entropy: the low-temperature monoclinic form is enthalpically favored, whereas the room-temperature orthorhombic form is entropically favored.

High-Temperature Deformation Processing Map Approach for Obtaining the Desired Microstructure in a Multi-component (Ni-Ti-Cu-Fe) Alloy

An equiatomic NiTiCuFe multi-component alloy with simple body-centered cubic (bcc) and face-centered cubic solid-solution phases in the microstructure was processed by vacuum induction melting furnace under dynamic Ar atmosphere. High-temperature uniaxial compression experiments were conducted on it in the temperature range of 1073 K to 1303 K (800 degrees C to 1030 degrees C) and strain rate range of 10(-3) to 10(-1) s(-1). The data generated were analyzed with the aid of the dynamic materials model through which power dissipation efficiency and instability maps were generated so as to identify the governing deformation mechanisms that are operative in different temperature-strain rate regimes with the aid of complementary microstructural analysis of the deformed specimens. Results indicate that the stable domain for the high temperature deformation of the multi-component alloy occurs in the temperature range of 1173 K to 1303 K (900 degrees C to 1030 degrees C) and (epsilon) over dot range of 10(-3) to 10(-1.2) s(-1), and the deformation is unstable at T = 1073 K to 1153 K (800 degrees C to 880 degrees C) and (epsilon) over dot = 10(-3) to 10(-1.4) s(-1) as well as T = 1223 K to 1293 K (950 degrees C to 1020 degrees C) and (epsilon) over dot = 10(-1.4) to 10(-1) s(-1), with adiabatic shear banding, localized plastic flow, or cracking being the unstable mechanisms. A constitutive equation that describes the flow stress of NiTiCuFe multi-component alloy as a function of strain rate and deformation temperature was also determined. (C) The Minerals, Metals & Materials Society and ASM International 2015

Conjugate Model for Heat and Mass Transfer of Porous Wall in the High Temperature Gas Flow

Heat and mass transfer of a porous permeable wall in a high temperature gas dynamical flow is considered. Numerical simulation is conducted on the ground of the conjugate mathematical model which includes filtration and heat transfer equations in a porous body and boundary layer equations on its surface. Such an approach enables one to take into account complex interaction between heat and mass transfer in the gasdynamical flow and in the structure subjected to this flow. The main attention is given to the impact of the intraporous heat transfer intensity on the transpiration cooling efficiency.

Electrothermal stress in conductive body with collinear cracks

The temperature and stress field in a thin plate with collinear cracks interrupting an electric current field are determined. This is accomplished by using a complex function method that allows a direct means of finding the distribution of the electric current, the temperature and stress field. Temperature dependency for the heat-transfer coefficient, coefficient of linear expansion and the elastic modulus are considered. As an example, temperature distribution is calculated for an alloy (No. GH2132) plate with two collinear cracks under high temperature. Relationships between the stress, temperature, electric density and crack length are obtained. Crack trajectories emanating from existing crack are predicted by application of the strain energy density criterion which can also be used for finding the load carrying capacity of the cracked plate. (C) 2003 Elsevier Ltd. All rights reserved.

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.

Ontogenetic patterns and temperature-dependent growth rates in early life stages of Pacific cod (Gadus macrocephalus)

Pacific cod (Gadus macrocephalus) is an important component of fisheries and food webs in the North Pacific Ocean and Bering Sea. However, vital rates of early life stages of this species have yet to be described in detail. We determined the thermal sensitivity of growth rates of embryos, preflexion and postflexion larvae, and postsettlement juveniles. Growth rates (length and mass) at each ontogenetic stage were measured in three replicate tanks at four to five temperatures. Nonlinear regression was used to obtain parameters for independent stage-specific growth functions and a unified size- and temperature-dependent growth function. Specific growth rates increased with temperature at all stages and generally decreased with increases in body size. However, these analyses revealed a departure from a strict size-based allometry in growth patterns, as reduced growth rates were observed among preflexion larvae: the reduction in specific growth rate between embryos and free-swimming larvae was greater than expected based on body size differences. Growth reductions in the preflexion larvae appear to be associated with increased metabolic rates and the transition from endogenous to exogenous feeding. In future studies, experiments should be integrated across life transitions to more clearly define intrinsic ontogenetic and size-dependent growth patterns because these are critical for evaluations of spatial and temporal variation in habitat quality.

Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana.

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent--disruption of one class does not affect the other--suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.

Influence of sex, spawning, starvation and water temperature on fatty acid composition in Tilapia mossambica

The influence of sex, spawning, starvation and water temperature on the fatty acid composition of Tilapia mossambica has been studied. Tilapia egg lipid was found to have unusually high percentage of C sub(22:6) fatty acids (9.09%) compared to body and intestinal lipids. The C sub(16:1) acid was much less in the egg lipids (3.5%) whereas it was 11% in the body lipids. There was no significant difference in the fatty acid composition of body and intestinal lipids of male and female tilapia. Starvation caused the presence of high content of lower fatty acids (C sub(6), C sub(8), C sub(30), C sub(12) and C sub(33)) in the body lipids. Water temperature also influenced the fatty acid composition of Tilapia; the difference was more significant in body lipids than in intestinal lipids.

Effects of preferential transport in turbulent bluff-body-stabilized lean premixed CH 4/air flames

Preferential species diffusion is known to have important effects on local flame structure in turbulent premixed flames, and differential diffusion of heat and mass can have significant effects on both local flame structure and global flame parameters, such as turbulent flame speed. However, models for turbulent premixed combustion normally assume that atomic mass fractions are conserved from reactants to fully burnt products. Experiments reported here indicate that this basic assumption may be incorrect for an important class of turbulent flames. Measurements of major species and temperature in the near field of turbulent, bluff-body stabilized, lean premixed methane-air flames (Le=0.98) reveal significant departures from expected conditional mean compositional structure in the combustion products as well as within the flame. Net increases exceeding 10% in the equivalence ratio and the carbon-to-hydrogen atom ratio are observed across the turbulent flame brush. Corresponding measurements across an unstrained laminar flame at similar equivalence ratio are in close agreement with calculations performed using Chemkin with the GRI 3.0 mechanism and multi-component transport, confirming accuracy of experimental techniques. Results suggest that the large effects observed in the turbulent bluff-body burner are cause by preferential transport of H 2 and H 2O through the preheat zone ahead of CO 2 and CO, followed by convective transport downstream and away from the local flame brush. This preferential transport effect increases with increasing velocity of reactants past the bluff body and is apparently amplified by the presence of a strong recirculation zone where excess CO 2 is accumulated. © 2011 The Combustion Institute.

Optimum temperature for the growth performance of juvenile orange-spotted grouper (Epinephelus coioides H.)

Effects of water temperature (17, 21, 25, 30 and 35 degrees C) and body size (14.75-281.41 g initial body weight) on food consumption, growth, feed conversion, and dry matter content in orange-spotted grouper fed to satiation were investigated. The combined effect of temperature (T, degrees C) and body weight (W, g) on maximum food consumption (C-max, g/day) was described as: InCmax= -7.411+0.828 lnW+0.317T-0.004 7T(2), and the optimum feeding temperature was 33.9 degrees C. The combined effect of temperature and body weight on growth (G) was described as: InG= -4.461-0.208lnW+0.394T-0.006 3T(2). The optimum growth temperature was 31.4 degrees C, whereas overall growth rates were high at 25, 30 and 35 degrees C. Feed conversion efficiencies (FCE, %), increasing first and then decreasing with increasing temperature, averaged from 1.8 to 2.1 in terms of dry weight of food fish. The optimum temperature for FCE tended to be lower than that for growth or feeding. Dry matter content increased with both increasing water temperature (17, 25, 30 and 35 degrees C) and body weight, and the combined effect of temperature and body weight on dry matter content (DM, %) was described as: lnDM =3.232+0.01 4 lnW-0.004 4T+0.001 2TlnW.

Calibration of alkenone unsaturation index with growth temperature for a lacustrine species, Chrysotila lamellosa (Haptophyceae)

Although long chain alkenones (LCKs) occur widely in lacustrine sediments, their origin is not clear. Here, we report a lacustrine source, the non-calcifying species Chrysotila lamellosa Anand (Haptophyceae), collected and isolated from an inland saline water body, Lake Xiarinur (Inner Mongolia, China). Its alketione pattern is similar to those of coastal marine strains of C lamellosa,but the relationship between U-37(K') index and culture temperature for the lacustrine species is quite different from that of the coastal species. A significant feature of the alkenones in this strain of C lamellosa is a lack of C-38 methyl alkenones, which might be used to distinguish the species from the marine haptophyte species Emiliania huxleyi and Gephyrocapsa oceanica. The higher C-38 tetraunsaturated compound abundance might be another important feature for distinguishing the C lamellosa alkenone producer from the coastal species Isochrysis galbana. This alkenone distribution pattern has been detected in many lakes, which suggests that C lamellosa or a closely related species might be a very common alkenone precursor in lacustrine systems. We examined U-37(K') and U-37(K) values for C lamellosa as a function of culture temperature in a batch culture experiment. The calibration for U-37(K') vs. culture temperature (T) was U-37(K') = 0.0011 x T-2 - 0.0157 x T + 0.1057(n = 14, r(2) = 0.99) from 10 degrees C to 22 degrees C or U-37(K') = 0.0257 x T - 0.2608(n = 9, r(2) = 0.97) from 14 degrees C to 22 degrees C. U-37(K) vs. culture temperature was U-37(K) = 0 0377 x T - 0.5992(n = 14, r(2) = 0.98) from 10 degrees C to 22 degrees C. Our experiments show that the alkenone unsaturation index (U-37(K')) is strongly controlled by culture temperature and can be used for palaeoclimate reconstruction. (C) 2007 Elsevier Ltd. All rights reserved.

Changes in the patterns of inorganic nitrogen and TN/TP ratio and the associated mechanism of biological regulation in the shallow lakes of the middle and lower reaches of the Yangtze River

The changes of NH3-N, NO3-N, NO2-N and TN/TP were studied during growth and non-growth season in 33 subtropical shallow lakes in the middle and lower reaches of the Yangtze River. There were significant positive correlations among all nutrient concentrations, and the correlations were better in growth season than in non-growth season. When TP > 0.1 mgL(-1), NH3-N increased sharply in non-growth season with increasing TP, and NO3-N increased in growth season but decreased in non-growth season with TP. These might be attributed to lower dissolved oxygen and low temperature in non-growth season of the hypereutrophic lakes, since nitrification is more sensitive to dissolved oxygen and temperature than anti nitrification. When 0.1 mgL(-1)> TP > 0.035 mgL(-1), TN and all kinds of inorganic nitrogen were lower in growth season than in non-growth season, and phytoplankton might be the vital regulating factor. When TP < 0.035 mgL(-1), inorganic nitrogen concentrations were relatively low and NH3-N, NO2-N had significant correlations with phytoplankton, indicating that NH3-N and NO2-N might be limiting factors to phytoplankton. In addition, TN/TP went down with decline in TIP concentration, and TN and inorganic nitrogen concentrations were obviously lower in growth season than in non-growth season, suggesting that decreasing nitrogen (especially NH3-N and NO3-N) was an important reason for the decreasing TN/TP in growth season. The ranges of TN/TP were closely related to trophic level in both growth and non-growth seasons, and it is apparent that in the eutrophic and hypertrophic state the TN/TP ratio was obviously lower in growth season than in non-growth season. The changes of the TN/TP ratio were closely correlated with trophic levels, and both declines of TN in the water column and TP release from the sediment were important factors for the decline of the TN/TP ratio in growth season.