Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO₂ ^– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are k _app1 = 9.6±0.2 M^–1 s^–1 and k _app2 = 1.2±0.1 M^–1 s^–1 (at pH 7.4 and 20°C). The k _app1 and k _app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h _app = 3.8×10⁴ M^–1 s^–1 and h ₀ = 2.8×10^–1 s^–1 (at pH 7.4 and 20°C). The pH-dependence of h _on and h ₀ values reflects the acid-base equilibrium of peroxynitrite (pK _a = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.     

The Final Frontier of pH and the Undiscovered Country Beyond

The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K _w = [H⁺]×[OH⁻].We used K _w [in a reversed way] to calculate the number of undissociated H₂O molecules required by this equilibrium constant to yield at least one of its daughter ions, H⁺ or OH⁻ at a given pH. In this way we obtained a formula that relates pH to the minimal volume V_pH required to provide a physical meaning to K _w, (where N _A is Avogadro’s number). For example, at pH 7 (neutral at 25°C) V_pH = 16.6 aL. Any deviation from neutral pH results in a larger V_pH value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H⁺ ions at equilibrium, thus the definition of pH based on K _w is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H⁺ ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H⁺ ions as a general source for biochemical reactions. Consequences of this finding are discussed.     

HCO3?-independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase

The mechanisms of HCO₃⁻-independent intracellular pH (pH_i) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pH_i in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H⁺ pump by Cl⁻ removal or via the selective inhibitor bafilomycin had only a small effect upon resting pH_i and recovery following an acid load. In contrast, resting pH_i in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pH_i recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO₃⁻-independent pH_i regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.     

Genetic and Phenotypic Correlations between Performance Traits with Meat Quality and Carcass Characteristics in Commercial Crossbred Pigs

Genetic correlations between performance traits with meat quality and carcass traits were estimated on 6,408 commercial crossbred pigs with performance traits recorded in production systems with 2,100 of them having meat quality and carcass measurements. Significant fixed effects (company, sex and batch), covariates (birth weight, cold carcass weight, and age), random effects (additive, litter and maternal) were fitted in the statistical models. A series of pairwise bivariate analyses were implemented in ASREML to estimate heritability, phenotypic, and genetic correlations between performance traits (n = 9) with meat quality (n = 25) and carcass (n = 19) traits. The animals had a pedigree compromised of 9,439 animals over 15 generations. Performance traits had low-to-moderate heritabilities (±SE), ranged from 0.07±0.13 to 0.45±0.07 for weaning weight, and ultrasound backfat depth, respectively. Genetic correlations between performance and carcass traits were moderate to high. The results indicate that: (a) selection for birth weight may increase drip loss, lightness of longissimus dorsi, and gluteus medius muscles but may reduce fat depth; (b) selection for nursery weight can be valuable for increasing both quantity and quality traits; (c) selection for increased daily gain may increase the carcass weight and most of the primal cuts. These findings suggest that deterioration of pork quality may have occurred over many generations through the selection for less backfat thickness, and feed efficiency, but selection for growth had no adverse effects on pork quality. Low-to-moderate heritabilities for performance traits indicate that they could be improved using traditional selection or genomic selection. The estimated genetic parameters for performance, carcass and meat quality traits may be incorporated into the breeding programs that emphasize product quality in these Canadian swine populations.     

Assessment of Genetic Correlation between Bacterial Cold Water Disease Resistance and Spleen Index in a Domesticated Population of Rainbow Trout: Identification of QTL on Chromosome Omy19

Selective breeding of animals for increased disease resistance is an effective strategy to reduce mortality in aquaculture. However, implementation of selective breeding programs is limited by an incomplete understanding of host resistance traits. We previously reported results of a rainbow trout selection program that demonstrated increased survival following challenge with Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD). Mechanistic study of disease resistance identified a positive phenotypic correlation between post-challenge survival and spleen somatic-index (SI). Herein, we investigated the hypothesis of a genetic correlation between the two traits influenced by colocalizing QTL. We evaluated the inheritance and calculated the genetic correlation in five year-classes of odd- and even-year breeding lines. A total of 322 pedigreed families (n = 25,369 fish) were measured for disease resistance, and 251 families (n = 5,645 fish) were evaluated for SI. Spleen index was moderately heritable in both even-year (h² = 0.56±0.18) and odd-year (h² = 0.60±0.15) lines. A significant genetic correlation between SI and BCWD resistance was observed in the even-year line (r_g = 0.45±0.20, P = 0.03) but not in the odd-year line (r_g = 0.16±0.12, P = 0.19). Complex segregation analyses of the even-year line provided evidence of genes with major effect on SI, and a genome scan of a single family, 2008132, detected three significant QTL on chromosomes Omy19, 16 and 5, in addition to ten suggestive QTL. A separate chromosome scan for disease resistance in family 2008132 identified a significant BCWD QTL on Omy19 that was associated with time to death and percent survival. In family 2008132, Omy19 microsatellite alleles that associated with higher disease resistance also associated with increased spleen size raising the hypothesis that closely linked QTL contribute to the correlation between these traits. To our knowledge, this is the first estimation of spleen size heritability and evidence for genetic linkage with specific disease resistance in a teleost fish.     

Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Root-Associated N2 Fixation (Acetylene Reduction) by Enterobacteriaceae and Azospirillum Strains in Cold-Climate Spodosols

N₂ fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C₂H₂ reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C₂H₄·g⁻¹ (dry weight)· h⁻¹ when incubated at pO₂ 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C₂H₄· g⁻¹ (dry weight)·h⁻¹ without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N₂-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

pH Tolerance in Freshwater Bacterioplankton: Trait Variation of the Community as Measured by Leucine Incorporation

pH is an important factor determining bacterial community composition in soil and water. We have directly determined the community tolerance (trait variation) to pH in communities from 22 lakes and streams ranging in pH from 4 to 9 using a growth-based method not relying on distinguishing between individual populations. The pH in the water samples was altered to up to 16 pH values, covering in situ pH ± 2.5 U, and the tolerance was assessed by measuring bacterial growth (Leu incorporation) instantaneously after pH adjustment. The resulting unimodal response curves, reflecting community tolerance to pH, were well modeled with a double logistic equation (mean R² = 0.97). The optimal pH for growth (pH_opt) among the bacterial communities was closely correlated with in situ pH, with a slope (0.89 ± 0.099) close to unity. The pH interval, in which growth was ≥90% of that at pH_opt, was 1.1 to 3 pH units wide (mean 2.0 pH units). Tolerance response curves of communities originating from circum-neutral pH were symmetrical, whereas in high-pH (8.9) and especially in low-pH (<5.5) waters, asymmetric tolerance curves were found. In low-pH waters, decreasing pH was more detrimental for bacterial growth than increasing pH, with a tendency for the opposite for high-pH waters. A pH tolerance index, using the ratio of growth at only two pH values (pH 4 and 8), was closely related to pH_opt (R² = 0.83), allowing for easy determination of pH tolerance during rapid changes in pH.     

Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals

Mitochondrial Ca²⁺ uptake exerts dual effects on mitochondria. Ca²⁺ accumulation in the mitochondrial matrix dissipates membrane potential (_ΔΨ_m), but Ca²⁺ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca²⁺ ([Ca²⁺]_m) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca²⁺]_m in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca²⁺ indicators with a 20-fold difference in Ca²⁺ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca²⁺ indicator (D4cpv) calibrated against known Ca²⁺ levels. Matrix pH (pH_m) and _ΔΨ_m were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca²⁺]_m was 0.22 ± 0.04 μM, but it rose to ∼26 μM (24.3 ± 3.4 μM with rhod-FF/rhod-5N and 27.0 ± 2.6 μM with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca²⁺]_m coincided with a rapid elevation in pH_m and was followed by an after-stimulus _ΔΨ_m hyperpolarization. However, pH_m decreased and no _ΔΨ_m hyperpolarization was observed in response to lower levels of [Ca²⁺]_m, up to 13.1 μM. These data indicate that surprisingly high levels of [Ca²⁺]_m are required to stimulate presynaptic mitochondrial energy metabolism.     

Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H⁺ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H⁺ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH_o) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH_i) and net H⁺ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH_i remained above 7.0 within a pH_o range of 4 to 6 and decreased below pH_o 4. Above pH_o 4, H⁺ extrusion increased correspondingly, with the maximum value at pH_o 5.5, and below pH_o 4, a net H⁺ influx was observed. Without glucose in the medium, the pH_i decreased, and a net H⁺ influx was observed below pH_o 5.5. A high correlation (R = 0.75 to 0.92) between the pH_i and net H⁺ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH_i homeostasis in L. monocytogenes subjected to acid stress.     

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases from Thermosyntropha lipolytica

Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH^25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH^25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C_18:1) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca²⁺ and one Mn²⁺ ion per monomeric enzyme unit. The addition of 1 mM MnCl₂ to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.     

Genome-Wide Association Study Identifies Loci and Candidate Genes for Body Composition and Meat Quality Traits in Beijing-You Chickens

Body composition and meat quality traits are important economic traits of chickens. The development of high-throughput genotyping platforms and relevant statistical methods have enabled genome-wide association studies in chickens. In order to identify molecular markers and candidate genes associated with body composition and meat quality traits, genome-wide association studies were conducted using the Illumina 60 K SNP Beadchip to genotype 724 Beijing-You chickens. For each bird, a total of 16 traits were measured, including carcass weight (CW), eviscerated weight (EW), dressing percentage, breast muscle weight (BrW) and percentage (BrP), thigh muscle weight and percentage, abdominal fat weight and percentage, dry matter and intramuscular fat contents of breast and thigh muscle, ultimate pH, and shear force of the pectoralis major muscle at 100 d of age. The SNPs that were significantly associated with the phenotypic traits were identified using both simple (GLM) and compressed mixed linear (MLM) models. For nine of ten body composition traits studied, SNPs showing genome wide significance (P<2.59E−6) have been identified. A consistent region on chicken (Gallus gallus) chromosome 4 (GGA4), including seven significant SNPs and four candidate genes (LCORL, LAP3, LDB2, TAPT1), were found to be associated with CW and EW. Another 0.65 Mb region on GGA3 for BrW and BrP was identified. After measuring the mRNA content in beast muscle for five genes located in this region, the changes in GJA1 expression were found to be consistent with that of breast muscle weight across development. It is highly possible that GJA1 is a functional gene for breast muscle development in chickens. For meat quality traits, several SNPs reaching suggestive association were identified and possible candidate genes with their functions were discussed.     

Disease Resistance in Atlantic Salmon (Salmo salar): Coinfection of the Intracellular Bacterial Pathogen Piscirickettsia salmonis and the Sea Louse Caligus rogercresseyi

Background

Naturally occurring coinfections of pathogens have been reported in salmonids, but their consequences on disease resistance are unclear. We hypothesized that 1) coinfection of Caligus rogercresseyi reduces the resistance of Atlantic salmon to Piscirickettsia salmonis; and 2) coinfection resistance is a heritable trait that does not correlate with resistance to a single infection.

Methodology

In total, 1,634 pedigreed Atlantic salmon were exposed to a single infection (SI) of P. salmonis (primary pathogen) or coinfection with C. rogercresseyi (secondary pathogen). Low and high level of coinfection were evaluated (LC = 44 copepodites per fish; HC = 88 copepodites per fish). Survival and quantitative genetic analyses were performed to determine the resistance to the single infection and coinfections.

Main Findings

C. rogercresseyi significantly increased the mortality in fish infected with P. salmonis (SI mortality = 251/545; LC mortality = 544/544 and HC mortality = 545/545). Heritability estimates for resistance to P. salmonis were similar and of medium magnitude in all treatments (h ² _SI = 0.23±0.07; h ² _LC = 0.17±0.08; h ² _HC = 0.24±0.07). A large and significant genetic correlation with regard to resistance was observed between coinfection treatments (r_g LC-HC = 0.99±0.01) but not between the single and coinfection treatments (r_g SI-LC = −0.14±0.33; r_g SI-HC = 0.32±0.34).

Conclusions/Significance

C. rogercresseyi, as a secondary pathogen, reduces the resistance of Atlantic salmon to the pathogen P. salmonis. Resistance to coinfection of Piscirickettsia salmonis and Caligus rogercresseyi in Atlantic salmon is a heritable trait. The absence of a genetic correlation between resistance to a single infection and resistance to coinfection indicates that different genes control these processes. Coinfection of different pathogens and resistance to coinfection needs to be considered in future research on salmon farming, selective breeding and conservation.     

A note on meat quality traits of pheasants (<Emphasis Type="Italic">Phasianus colchicus</Emphasis>)

Meat yield, proximate composition, pH and drip loss of breast and thigh muscles were studied in 29 hunted male and 32 slaughtered female pheasants. In the breast muscles of additional 14 hunted male pheasants, colour, cooking loss and shear force were measured. Weight of muscles of hunted male pheasants was higher than that of slaughtered females, but the percentages of breast and thigh muscles relative to the dressed carcass did not differ. Average protein concentrations in lean muscles were above 22%, and average fat was below 1.2%. In breast muscles, pH₂₄ was lower than in thigh (ca. 5.6 vs. 6.0), and, consequently, drip loss was higher (2.2-3.0% vs. 1.0-1.5%). Colour of breast muscles was characterised by L*a*b* values of about 55, 4 and 8-9, respectively. The shear force of breast muscles was about 30 N/cm².     

Post-Prandial Protein Handling: You Are What You Just Ate

Background

Protein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults.

Objective

To provide a comprehensive overview of post-prandial protein handling, ranging from dietary protein digestion and amino acid absorption, the uptake of dietary protein derived amino acids over the leg, the post-prandial stimulation of muscle protein synthesis rates, to the incorporation of dietary protein derived amino acids in de novo muscle protein.

Design

12 healthy young males ingested 20 g intrinsically [1-¹³C]-phenylalanine labeled protein. In addition, primed continuous L-[ring-²H₅]-phenylalanine, L-[ring-²H₂]-tyrosine, and L-[1-¹³C]-leucine infusions were applied, with frequent collection of arterial and venous blood samples, and muscle biopsies throughout a 5 h post-prandial period. Dietary protein digestion, amino acid absorption, splanchnic amino acid extraction, amino acid uptake over the leg, and subsequent muscle protein synthesis were measured within a single in vivo human experiment.

Results

55.3±2.7% of the protein-derived phenylalanine was released in the circulation during the 5 h post-prandial period. The post-prandial rise in plasma essential amino acid availability improved leg muscle protein balance (from -291±72 to 103±66 μM·min^-1·100 mL leg volume^-1; P<0.001). Muscle protein synthesis rates increased significantly following protein ingestion (0.029±0.002 vs 0.044±0.004%·h^-1 based upon the muscle protein bound L-[ring-²H₅]-phenylalanine enrichments (P<0.01)), with substantial incorporation of dietary protein derived L-[1-¹³C]-phenylalanine into de novo muscle protein (from 0 to 0.0201±0.0025 MPE).

Conclusion

Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, thereby improving whole-body and leg protein balance. About 20% of the dietary protein derived amino acids released in the circulation are taken up in skeletal muscle tissue following protein ingestion, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein synthesis.

Trial Registration

trialregister.nl 3638     

Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes

Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp-PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pH_v) of 4.74 ± 0.1 is generated in the presence of 0.1 millimolar PP_i, but only 4.93 ± 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PP_i. Cl⁻-stimulates the H⁺-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K_½ ≈ 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PP_i. The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pH_v unit has been estimated. From the free energy (−11.42 kilojoules per mole) for the hydrolysis of PP_i under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H⁺ per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H⁺-pump rather than as a pyrophosphate synthetase.     

Cross-Talk in Mechanomyographic Signals from the Forearm Muscles during Sub-Maximal to Maximal Isometric Grip Force

Purpose

This study aimed: i) to examine the relationship between the magnitude of cross-talk in mechanomyographic (MMG) signals generated by the extensor digitorum (ED), extensor carpi ulnaris (ECU), and flexor carpi ulnaris (FCU) muscles with the sub-maximal to maximal isometric grip force, and with the anthropometric parameters of the forearm, and ii) to quantify the distribution of the cross-talk in the MMG signal to determine if it appears due to the signal component of intramuscular pressure waves produced by the muscle fibers geometrical changes or due to the limb tremor.

Methods

Twenty, right-handed healthy men (mean ± SD: age  = 26.7±3.83 y; height  = 174.47±6.3 cm; mass  = 72.79±14.36 kg) performed isometric muscle actions in 20% increment from 20% to 100% of the maximum voluntary isometric contraction (MVIC). During each muscle action, MMG signals generated by each muscle were detected using three separate accelerometers. The peak cross-correlations were used to quantify the cross-talk between two muscles.

Results

The magnitude of cross-talk in the MMG signals among the muscle groups ranged from, R²_{x, y} = 2.45–62.28%. Linear regression analysis showed that the magnitude of cross-talk increased linearly (r² = 0.857–0.90) with the levels of grip force for all the muscle groups. The amount of cross-talk showed weak positive and negative correlations (r² = 0.016–0.216) with the circumference and length of the forearm respectively, between the muscles at 100% MVIC. The cross-talk values significantly differed among the MMG signals due to: limb tremor (MMG_TF), slow firing motor unit fibers (MMG_SF) and fast firing motor unit fibers (MMG_FF) between the muscles at 100% MVIC (p<0.05, η ² = 0.47–0.80).

Significance

The results of this study may be used to improve our understanding of the mechanics of the forearm muscles during different levels of the grip force.     

Growth Kinetics and Yield Coefficients of the Extreme Thermophile Thermothrix thiopara in Continuous Culture

Thermothrix thiopara did not appear to be stressed at high temperature (72°C). Both the actual and theoretical yields were higher than those of analogous mesophilic sulfur bacteria, and the specific growth rate (μ_max) was more rapid than that of most autotrophs. The specific growth rate (0.58 h⁻¹), specific maintenance rate (0.11 h⁻¹), actual molar growth yield at μ_max (Y_max = 16 g mol⁻¹), and theoretical molar growth yield (Y_G = 24 g mol⁻¹) were all higher for T. thiopara (72°C) than for mesophilic (25 to 30°C) Thiobacillus spp. The growth efficiencies for T. thiopara at 70 and 75°C (0.84 and 0.78) were significantly higher than at 65°C (0.47). Corresponding specific maintenance rates were highest at 65°C (0.41 h⁻¹) and lowest at 70 and 75°C (0.11 and 0.15 h⁻¹, respectively). Growth efficiencies of metabolically similar mesophiles were generally higher than for T. thiopara. However, the actual yields at μ_max were higher for T. thiopara because its theoretical yield was higher. Thus, at 70°C, T. thiopara was capable of deriving more metabolically useful energy from thiosulfate than were mesophilic sulfur bacteria at 25 and 30°C. The low growth efficiency of T. thiopara reflected higher maintenance expenditures. T. thiopara had higher maintenance rates than Thiobacillus ferroxidans or Thiobacillus denitrificans, but also attained higher molar growth yields. It is concluded that sulfur metabolism may be more efficient overall at extremely high temperatures due to increased theoretical yields despite increased maintenance requirements.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.