Population-Referenced Percentiles for Waist-Worn Accelerometer-Derived Total Activity Counts in U.S. Youth: 2003 – 2006 NHANES

Background

The total activity volume performed is an overall measure that takes into account the frequency, intensity, and duration of activities performed. The importance of considering total activity volume is shown by recent studies indicating that light physical activity (LPA) and intermittent moderate-to-vigorous physical activity (MVPA) have health benefits. Accelerometer-derived total activity counts (TAC) per day from a waist-worn accelerometer can serve as a proxy for an individual''s total activity volume. The purpose of this study was to develop age- and gender-specific percentiles for daily TAC, minutes of MVPA, and minutes of LPA in U.S. youth ages 6 – 19 y.

Methods

Data from the 2003 – 2006 NHANES waist-worn accelerometer component were used in this analysis. The sample was composed of youth aged 6 – 19 years with at least 4 d of ≥ 10 hours of accelerometer wear time (N = 3698). MVPA was defined using age specific cutpoints as the total number of minutes at ≥4 metabolic equivalents (METs) for youth 6 – 17 y or minutes with ≥2020 counts for youth 18 – 19 y. LPA was defined as the total number of minutes between 100 counts and the MVPA threshold. TAC/d, MVPA, and LPA were averaged across all valid days.

Results

For males in the 50^th percentile, the median activity level was 441,431 TAC/d, with 53 min/d of MVPA and 368 min/d of LPA. The median level of activity for females was 234,322 TAC/d, with 32 min/d of MVPA and 355 min/d of LPA.

Conclusion

Population referenced TAC/d percentiles for U.S. youth ages 6-19 y provide a novel means of characterizing the total activity volume performed by children and adolescents.     

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.¹ TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.² The murine TAC model was first validated by Rockman et al.¹, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.^{3, 4} With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice. Download video file.^{(94M, mp4)}     

Tlr4 Deficiency Protects against Cardiac Pressure Overload Induced Hyperinflammation

Transverse aortic constriction provokes a pro-inflammatory reaction and results in cardiac hypertrophy. Endogenous ligands contribute to cardiac hypertrophy via toll-like receptor (TLR)-4 binding. A lack of TLR4 signaling diminishes hypertrophy and inflammation. Wild type mice undergoing aortic constriction respond to a lipopolysaccharide second-hit stimulus with hyperinflammation. The objective of this study was to assess whether other second-hit challenges utilizing TLR ligands provoke a comparable inflammatory reaction, and to find out whether this response is absent in TLR4 deficient mice. Assuming that cardiac stress alters the expression of pattern recognition receptors we analyzed the effects of transverse aortic constriction and second-hit virulence factor treatment on TLR expression, as well as cytokine regulation. Wild type and Tlr4 ^-/- mice were subjected to three days of TAC and subsequently confronted with gram-positive TLR2 ligand lipoteichoic acid (LTA, 15mg/g bodyweight) or synthetic CpG-oligodesoxynucleotide 1668 thioate (20 nmol/kg bodyweight, 30 min after D-galactosamin desensitization) signaling via TLR9. Hemodynamic measurements and organ preservation were performed 6 h after stimulation. Indeed, the study revealed a robust enhancement of LTA induced pattern recognition receptor and cytokine mRNA expression and a LTA-dependent reduction of hemodynamic pressure in TAC wild type mice. Second-Hit treatment with CpG-ODNs led to similar results. However, second-hit effects were abolished in Tlr4 ^-/- mice. In total, these data indicate for the first time that cardiac stress increases the inflammatory response towards both, gram-negative and gram-positive, TLR ligands as well as bacterial DNA. The decrease of the inflammatory response upon TLR2 and -9 ligand challenge in TAC Tlr4 ^-/- mice demonstrates that a lack of TLR4 signaling does not only prevent left ventricular hypertrophy but also protects the mice from a cardiac stress induced hyperinflammatory reaction.     

AKINETE FORMATION IN PLANKTONIC ANABAENA SPP. (CYANOBACTEFUA) BY TREATMENT WITH LOW TEMPERATURE1

The effects of temperature, light intensity and nutrient depletion on akinete formation in seven strains of planktonic Anabaena spp.: A. mucosa TAC426; A. crassa TAC436; A. spiroides TAC443 and TAC444; A. flosaquae TAC446; and A. ucrainica TAC448 and TAC449 were examined. A Marked Pfft of temperature on akinete formation was observed at 40 μmol photons·m^?2·sec^?1 and nutrient-sufficient conditions. At 20° C, akinetes did not develop in A. mucosa TAC426, A. crassa TAC436, A. spiroides TAC443, A. flos-aquae TAC446, or A. ucrainica TAC449 but were formed at frequencies of a little over 11% (ratio of filaments with akinetes to total filaments) in A. spiroides TAC444 and A. ucrainica TAC448. None of the strains fmd akinetes or heterocysts at 30° C and 35° C. At lower temperature (10° C and 15° C), akinetes developed in all the strains at maximum frequencies of 13.4–77.4% during the late exponential phase or late exponential to stationary phases of growth. With only one exception, low light or nutrient deletion did not lead to the induction of akinete diferentiation at 20° C. Only akinete formation in A. flosaquae TAC446 was induced by nitrogen deletion with a frequency of 12.1%, similar to that induced by low temperature, but the initiation of akinete formation in the strain was delayed compared to treatment with low temperature. These results show that temperature was the most important environmental factor triggering akinete formation in these species. In A. crassa TAC436 and A. spiroides TAC443 and TAC444, akinetes developed during the late exponential growth phase even though heterocysts were formed at a 100% frequency (ratio of filaments with heterocysts to total filaments) throughout the entire growth phase. In A. mucosa TAC426, A. flos-aquae TAC446, and A. ucrainica TAC448 and TAC449, there was a positive correlation between heterocyst and akinete formation, suggesting that the presence of a heterocyst may play a role in akinete formation.     

Picomole assay for N-methylhistidine by gas chromatography—Mass spectrometry

A simple, sensitive method for measuring N^τ-methylhistidine in biological samples using a deuterated internal standard and methane chemical ionization gas chromatography-mass spectrometry is described. After sample preparation, a single analysis can be completed in 3 min; analysis in duplicate, including sample preparation for 40 samples, can be completed at a rate of 15 min per sample. Nanomole amounts of N^τ-mmethylhistidine in urine or plasma samples are determined with a precision of 0.5%. Picomole amounts, released during in vitro rat epitrochlaris muscle incubations, are measured with a precision of 10%.     

Differential Effects of Tacrolimus versus Sirolimus on the Proliferation,Activation and Differentiation of Human B Cells

The direct effect of immunosuppressive drugs calcineurin inhibitor (Tacrolimus, TAC) and mTOR inhibitor (Sirolimus, SRL) on B cell activation, differentiation and proliferation is not well documented. Purified human B cells from healthy volunteers were stimulated through the B Cell Receptor with Anti-IgM + anti-CD40 + IL21 in the absence / presence of TAC or SRL. A variety of parameters of B cell activity including activation, differentiation, cytokine productions and proliferation were monitored by flow cytometry. SRL at clinically relevant concentrations (6 ng/ml) profoundly inhibited CD19⁺ B cell proliferation compared to controls whereas TAC at similar concentrations had a minimal effect. CD27⁺ memory B cells were affected more by SRL than naïve CD27^- B cells. SRL effectively blocked B cell differentiation into plasma cells (CD19⁺CD138⁺ and Blimp1⁺/Pax5^low cells) even at low dose (2 ng/ml), and totally eliminated them at 6 ng/ml. SRL decreased absolute B cell counts, but the residual responding cells acquired an activated phenotype (CD25⁺/CD69⁺) and increased the expression of HLA-DR. SRL-treated stimulated B cells on a per cell basis were able to enhance the proliferation of allogeneic CD4⁺CD25⁻ T cells and induce a shift toward the Th1 phenotype. Thus, SRL and TAC have different effects on B lymphocytes. These data may provide insights into the clinical use of these two agents in recipients of solid organ transplants.     

Quantitative determination of BMS186716, a thiol compound,in dog plasma by high-performance liquid chromatography–positive ion electrospray mass spectrometry after formation of the methyl acrylate adduct

As it is extremely unstable in blood, the thiol compound BMS186716 was stabilized by the addition of methyl acrylate (MA) to blood samples. The blood samples were then kept in ice for 10–15 min for completion of the Michael addition reaction to occur between the thiol group of BMS186716 and MA, after which the plasma was separated by centrifugation under refrigeration. For sample analysis, the standard and quality control samples were prepared by spiking blank plasma with the BMS186716–MA adduct. After addition of the internal standard, BMS188035–MA, each sample was acidified with HCl and then extracted with methyl tert.-butyl ether. Each reconstituted extract was injected into a high-performance liquid chromatography–positive ion electrospray ionization mass spectrometric system. The electrospray condition was chosen to enhance the [M+NH₄]⁺ signal at the expense of the [M+H]⁺ signal. Monitoring the [M+NH₄]⁺ signal, a lower limit of quantitation of 2.5 ng/ml was achieved, with 0.5 ml plasma. We have thus shown that a sulfhydryl compound (BMS186716) in blood can successfully be stabilized by reacting it with MA and that the adduct produced is adequately stable in blood and plasma to allow the development of a rugged quantitative bioanalytical method.     

Development of a B-cell maturation antigen-specific T-cell antigen coupler receptor for multiple myeloma

Background aimsT cells engineered with synthetic receptors have delivered powerful therapeutic results for patients with relapsed/refractory hematologic malignancies. The authors have recently described the T-cell antigen coupler (TAC) receptor, which co-opts the endogenous T-cell receptor (TCR) and activates engineered T cells in an HLA-independent manner. Here the authors describe the evolution of a next-generation TAC receptor with a focus on developing a TAC-engineered T cell for multiple myeloma.MethodsTo optimize the TAC scaffold, the authors employed a bona fide antigen-binding domain derived from the B-cell maturation antigen-specific monoclonal antibody C11D5.3, which has been used successfully in the clinic. The authors first tested humanized versions of the UCHT1 domain, which is used by the TAC to co-opt the TCR. The authors further discovered that the signal peptide affected surface expression of the TAC receptor. Higher density of the TAC receptor enhanced target binding in vitro, which translated into higher levels of Lck at the immunological synapse and stronger proliferation when only receptor–ligand interactions were present.ResultsThe authors observed that the humanized UCHT1 improved surface expression and in vivo efficacy. Using TAC T cells derived from both healthy donors and multiple myeloma patients, the authors determined that despite the influence of receptor density on early activation events and effector function, receptor density did not impact late effector functions in vitro, nor did the receptor density affect in vivo efficacy.ConclusionsThe modifications to the TAC scaffold described herein represent an important step in the evolution of this technology, which tolerates a range of expression levels without impacting therapeutic efficacy.     

Optimisation of phenolic compounds and antioxidant activity extraction conditions of a roasted mix of Tetrapleura tetraptera (Schumach & Thonn.) and Aframomum citratum (C. Pereira) fruits using response surface methodology (RSM)

The therapeutic abilities of Tetrapleura tetraptera and Aframomum citratum fruits used as spices are attributed to their bioactive molecules, including polyphenols. Sometimes used together and heated, they can undergo denaturation. The aim of the current study is to optimize the extraction of phenolic compounds and antioxidant potential of a roasted mix of Tetrapleura tetraptera and Aframomum citratum (95/5: w/w) fruits using RSM in a home food consumption context. The mix of spices was chosen according to the highest content of TPP and preliminary studies were performed to select the influencing variables. Roasting temperatures (130–170 °C), roasting times (10–15 min) and brewing times (8–15 min) were investigated with a rotatable central composite design. Experimental results were fitted to the second-order polynomial model where multiple regressions and ANOVA were used to determine the coefficients of the model and the optimal conditions for the considered responses. The two spices are good sources of phenolic compounds, and they also show significant (p < 0.05) dose-dependent radical scavenging activities (DPPH assay and inhibition of β-carotene discoloration) and reductive activities (FRAP assay and Phosphomolybdenum method). They significantly inhibit bovine serum albumin and 5-LOX denaturation. Brewing time and roasting time significantly (p < 0.05) influence the responses and there is a strong (R² = 0.93) correlation between the TPP and TAC of the beverage. The quadratic model fit well and the different factors used to test its accuracy and fitness were in satisfactory ranges. For TPP extraction (38.90 mgGAE/g dw) and TAC (50.75 mg TE/g dw) expression, the optimal conditions were reached at a roasting temperature of 150 °C, roasting time of 12.62 min, brewing time of 11.91 min and a desirability of 0.95. The novel information on the optimisation of the process can be further used by scientists, consumers and herbalists for effective handling of fruits during the extraction process.     

Isolation and characterization of plasma membrane fromAcanthamoeba culbertsoni

A rapid method for preparation of plasma membrane fromAcanthamoeba culbertsoni involving toluene treatment followed by lithium bromide extraction is described. In the plasma membrane preparation, 5′-nucleotidase, Na⁺ + K⁺ -ATPase, Mg²⁺ -ATPase and glucose-6-phosphatase activities were enriched. The membrane preparation was free from nucleic acid, cytochrome P-450 and cytochrome b5. Amino acid (¹⁴C-Ieucine) was not incorporated in the plasma membrane in 2 min. Succinic dehydrogenase was not detectable in the plasma membrane preparation. The molar ratio of cholesterol and phospholipids was 0.95 which is characteristics for plasma membranes. Under electronmicroscopy the preparation was homogenous without any other component of the cell. Plasma membrane proteins and glycoproteins were separated on acrylamide gel electrophoresis     

Preliminary Study on High-level Expression of Tandem-Arranged Tachyplesin-Encoding Gene in Bacillus subtilis Wb800 and its Antibacterial Activity

To produce tachyplesin, an antimicrobial peptide, by a stable and efficient gene engineering approach, cDNAs containing single tachyplesin gene sequence (tac)¹ and tandem repeat of tachyplesin gene sequence (tac2) were respectively developed by annealing two synthesized complementary single-stranded DNAs and constructed into pSBPTQ shuttle vector under the control of the SacB.p.s promoter. The vectors containing the target gene sequence were then transformed into Bacillus subtilis WB800, respectively. Both expression of tac and tac2 were induced by 2% sucrose. The fermentation supernatant was purified by regenerated cellulose membrane tubing (MWCO 2000) and the secreted TAC² and TAC2 were about 5 and 10 mg/l of supernatant, respectively. The antimicrobial activities of TAC and TAC2 were measured by the size of bacteriostatic circle of the fermentation supernatants against Escherichia coli K88. Ultrastructural alteration of E. coli K88 and Salmonella typhimurium was observed under scanning electron microscope and transmission electron microscopy. The results showed that in comparison with TAC, TAC2 was expressed at a higher level and also indicating strong antimicrobial activity both in vitro and in vivo. ¹ tac is tachyplesin gene; tac2 is a tandem repeat tachyplesin gene.²TAC is the expression product of tac; TAC2 is the expression product of tac2.     

Attenuation of cardiac hypertrophy by G‐CSF is associated with enhanced migration of bone marrow‐derived cells

Granulocyte‐colony stimulating factor (G‐CSF) has been shown to promote mobilization of bone marrow‐derived stem cells (BMCs) into the bloodstream associated with improved survival and cardiac function after myocardial infarction. Therefore, the aim of the present study was to investigate whether G‐CSF is able to attenuate cardiac remodelling in a mouse model of pressure‐induced LV hypertrophy focusing on mobilization and migration of BMCs. LV hypertrophy was induced by transverse aortic constriction (TAC) in C57BL/6J mice. Four weeks after TAC procedure. Mice were treated with G‐CSF (100 μg/kg/day; Amgen Biologicals) for 2 weeks. The number of migrated BMCs in the heart was analysed by flow cytometry. mRNA expression and protein level of different growth factors in the myocardium were investigated by RT‐PCR and ELISA. Functional analyses assessed by echocardiography and immunohistochemical analysis were performed 8 weeks after TAC procedure. G‐CSF‐treated animals revealed enhanced homing of VLA‐4⁺ and c‐kit⁺ BMCs associated with increased mRNA expression and protein level of the corresponding homing factors Vascular cell adhesion protein 1 and Stem cell factor in the hypertrophic myocardium. Functionally, G‐CSF significantly preserved LV function after TAC procedure, which was associated with a significantly reduced area of fibrosis compared to control animals. Furthermore, G‐CSF‐treated animals revealed a significant improvement of survival after TAC procedure. In summary, G‐CSF treatment preserves cardiac function and is able to diminish cardiac fibrosis after induction of LV hypertrophy associated with increased homing of VLA‐4⁺ and c‐kit⁺ BMCs and enhanced expression of their respective homing factors VCAM‐1 and SCF.     

Labelling of leukocytes with 99mTc-HMPAO for scintigraphy of inflammatory lesions and abscesses

A simplified and efficient procedure for ^99mTc-HMPAO-labelling of leukocytes is described. For this purpose, the pH and concentration of the ^99mTc-HMPAO preparation was modified. Leukocytes were isolated from a 20 mL mixture of patient blood, 5 mL ACD and 0.8 mL methylcellulose after 1 h sedimentation of erythrocytes and centrifugation (at 400 g) of the obtained plasma layer. Simultaneously, ^99mTc-HMPAO was prepared (one single-dose kit for two patients) by adding 2.2 mL ^99mTc-generator eluate and, after 10 min, 0.3 mL of phosphate buffer to lower the pH to 7. The isolated WBCs were then labelled by the addition of 1-1.2 mL of ^99mTc-HMPAO solution and incubated for 20 min. The unbound tracer was then discarded, the labelled WBC washed and finally resuspended in autologous cell-free plasma. Leukocytes labelled by this procedure were used for scintigraphic localization of inflammatory lesions and abscesses in the gastro-intestinal tract.The labelling efficiency was 60 ± 9%, with a separation yield of 55 ± 11%.     

Vasoactive intestinal polypeptide and endometrial blood flow in the goat

Endometrial blood flow was measured in seven goats twice before, once during, and twice after the close intra-arterial infusion of 300 pmol/min of vasoactive intestinal polypeptide (VIP). A saline solution of ⁸⁵krypton was injected intra-arterially and local blood flow calculated from the disappearance rate of the isotope from tissue surrounding a miniature Geiger-Müller probe in the uterine lumen. Initial blood flows (median and range) in animals pretreated with oestradiol-17β (N = 4) or with a regimen of oestradiol-17β and progesterone (N = 3), respectively, were 0.21 (0.11–0.35) and 0.20 (0.06–0.27) ml min^?1 g^?1. No significant change in endometrial blood flow occurred during or after the infusion of VIP, although the dose was sufficient to increase heart rate from 2.14 ± 0.15 to 2.32 ± 0.16 Hz (P < 0.001). The results indicate that VIP, in a dose known to increase myometrial blood flow in goats, is unable to evoke vasodilatation in the endometrium of this species. Since there is a corresponding variation in the density of nerve fibers containing VIP, it is suggested that VIP may play a role in regulating the partition of uterine blood flow in goats.     

Effect of maximal-intensity exercise on systemic nitro-oxidative stress in men and women

Objectives: The aim of this study was to test the hypotheses: (1) there is a negative correlation between protein and lipid oxidative damage following maximal-intensity exercise, and oxygen uptake and work intensity (%VO_2max) at the respiratory compensation point (RCP) in women and men; (2) nitro-oxidative stress following maximal-intensity exercise results from the intensification of anaerobic processes and muscle fibre micro-damage.

Methods: Study participants comprised 20 women (21.34±1.57 years) and 20 men (21.97±1.41 years) who performed a treadmill incremental test (IT); VO_2max: 45.08?±?0.91 and 57.38?±?1.22?mL?kg^?1?min^?1 for women and men, respectively. The oxidized low-density lipoprotein (ox-LDL), 3-nitrotyrosine (3-NT) concentration and creatine kinase (CK) as well as lactate dehydrogenase (LDH) activity were measured in the blood serum, and total antioxidative capacity (TAC) and lactate concentration (Lac) were determined in blood plasma before and after IT.

Results: After the IT, increases in ox-LDL, 3-NT, CK, and LDH were seen in both groups (P?P?P?Conclusions: The gain of ox-LDL and 3-NT following maximal-intensity exercise is independent of VO_2max, oxygen consumption and exercise intensity at RCP. This increase of ox-LDL and 3-NT is indicative of similar lipid and protein damage in women and men. A significant increase in TAC in women following maximal-intensity exercise is the result of muscle fibre micro-injuries.     

18F Labeled benzimidazole derivatives as potential radiotracer for positron emission tomography (PET) tumor imaging

This article reported the synthesis and bioevaluation of two [¹⁸F] labeled benzimidazole derivatives, 4-(5-(2-[¹⁸F] fluoro-4-nitrobenzamido)-1-methyl-1H-benzimidazol-2-yl) butanoic acid ([¹⁸F] FNBMBBA, [¹⁸F]a1) and 3-(2-fluoroethyl)-7-methyl-2-propyl-3H-benzimidazole-5-carboxylic acid ([¹⁸F] FEMPBBA, [¹⁸F]b1) for PET tumor imaging. The preparation [¹⁸F] FEMPBBA was completed in 1 h with overall radiochemical yield of 50–60% (without decay corrected). Biodistribution assay in S180 tumor bearing mice of both compounds were carried out, and the results are both meaningful. [¹⁸F] FEMPBBA which can be taken as a revision of [¹⁸F] FNBMBBA got an excellent result, and has significant advantages in some aspects compared with L-[¹⁸F] FET and [¹⁸F]-FDG in the same animal model, especially in tumor/brain uptake ratio. The tumor/brain uptake ratio of [¹⁸F] FEMPBBA gets to 4.81, 7.15, and 9.8 at 30 min, 60 min and 120 min, and is much higher than that of L-[¹⁸F] FET (2.54, 2.92 and 2.95) and [¹⁸F]-FDG (0.61, 1.02, 1.33) at the same time point. The tumor/muscle and tumor/blood uptake ratio of [¹⁸F] FEMPBBA is also higher than that of L-[¹⁸F] FET at 30 min and 60 min. This result indicates compound [¹⁸F] FEMPBBA is a promising radiotracer for PET tumor imaging.     

Determinants of the disposition of 14C-delta 9-tetrahydrocannabinol and 3H-delta 9-tetrahydrocannabinol.

Intragastric (i.g.) administration of ¹⁴C-THC plus ³H-THC to male Wistar and Fischer rats resulted in a more rapid disappearance of ¹⁴C than ³H from fresh blood or plasma. The concentrations of the two isotopes were equivalent from 1–4 hours when blood was analyzed after being dried. For each rat strain, apparent absorption of both isotopes was more rapid in fed than fasted rats and in young (130–150g) compared to older (250–260g) animals. The concentrations of ³H were significantly higher than ¹⁴C in the major organs which were analyzed fresh at 4 and 24 hours after drug administration, but the isotope levels were not different when the tissues were analyzed after lyophilization. Experiments indicate that tritiated water was produced metabolically from ³H-THC and was lost from blood and organs upon drying.The fresh blood levels of total ¹⁴C and unchanged ¹⁴C-THC were higher than total ³H and ³H-THC, respectively, from 40 min to 4 hrs following i.v. injection of ¹⁴C-THC plus ³H-THC in bile duct-cannulated rats. Similarly, the amount of ¹⁴C was higher than the amount of ³H in the urine. However, the concentration of ³H was higher than ¹⁴C in the bile after 20 min. The ³H level was higher than ¹⁴C at 4 hrs in the brain but lower than ¹⁴C in the liver, heart and spleen. Drying of body fluids and tissues before isotope analysis did not alter these results. It is concluded that the formation of tritiated water occurs in the gut after i.g. ³H-THC administration, and that the dispositions of ¹⁴C-THC and ³H-THC are not entirely equivalent following i.v. injection.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca²⁺-dependent ATPase and are also able to accumulate up to 10 μmol Ca²⁺ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a₃ and an increase in the content of [³²P]phosphoenzyme. The basic components in “calcium-oxalate preparation” from hearts are proteins with molecular weights of about 100 000 (Ca²⁺-dependent ATPase) and 55 000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca²⁺-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12–16 μmol P_i/min per mg protein. Enzyme activity of purified Ca²⁺-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca²⁺-dependent ATPases isolated from heart and skeletal muscles.     

Study of laccase production by Pleurotus ostreatus in a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasma

Aims: To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma. Methods and Results: Laccase production by P. ostreatus studied in a benchtop bioreactor was as high as, 874·0 U ml^?1 in presence of copper sulfate. The enzyme was used to replace metmyoglobin and hydrogen peroxide for the estimation of TAC in human plasma. The trolox equivalent antioxidant concentrations determined by the laccase‐based method and metmyoglobin method ranged from 1·63 ± 0·011 to 1·80 ± 0·006 mmol l^?1 and from 1·41 ± 0·004 to 1·51 ± 0·008 mmol l^?1 plasma, respectively. Conclusions: Pleurotus ostreatus produced high amount of extracellular laccase in a benchtop bioreactor. The enzyme can be used to assay TAC of blood plasma without the interference encountered with the hydrogen peroxide and metmyoglobin mediated assay method. Significance and Impact of the Study: Laccase production by P. ostreatus obtained in this study was the highest among all reported laccase producing white‐rot fungi. Moreover, an accurate laccase‐based assay method was developed for detection of TAC in human plasma.