The nature of post-translational formation of MM creatine kinase isoforms

Isoforms (derived from the same isoenzyme but distinguished by differences in isoelectric point) of MM creatine kinase appear in plasma after myocardial infarction. They are formed by conversion of the tissue form of creatine kinase (MM-A, pI 7.80) to progressively more acidic species (MM-B, pI 7.50) and MM-C (pI 7.20) after release into the circulation. To define the changes responsible for myocardial MM creatine kinase isoform formation in humans and dogs, purified isoforms were treated with trypsin or cyanogen bromide. The digests were fractionated by reverse-phase high pressure liquid chromatography. Comparison of proteolytic maps showed that MM-A and MM-C were each characterized by a single, unique peptide peak. Maps of MM-B creatine kinase contained both of these peaks. Sequence analysis and comparison with the complete amino acid sequence of MM creatine kinase showed that the peptide unique to MM-A corresponded to the COOH-terminal tryptic or CNBr peptide. The peptide unique to MM-C was shown to have the same amino acid composition except for lysine (the COOH-terminal amino acid). Thus, isoform formation is characterized by the successive removal of the COOH-terminal lysine residue from one M subunit at a time resulting in the conversion of MM-A to isoforms MM-B and MM-C.     

Analysis of plasmid samples on a microchip

We have developed a LabChip-based plasmid assay that runs on the Agilent 2100 Bioanalyzer. The assay determines the sizes and relative concentrations of the multiple forms of plasmid samples. Twelve samples can be analyzed on each chip in an automated run lasting approximately 30min. By using a supercoiled DNA sizing standard of 2-16kb, the size of the analyzed plasmid can be determined. The resulting MW has a relative standard deviation (CV) <5% and error <5%. Plasmids from 2-8kb can be separated with resolution better than 1kb. Topological isoforms in a plasmid sample can also be separated. However, due to differential staining, the heterogeneity of plasmid samples can only be measured if the signal of each isomer peak can be calibrated with pure standards for every isomer form. For a typical plasmid preparation which predominately is in the supercoiled form, the normalized corrected peak area for the supercoiled form correlates with the plasmid concentration in a broad range of 1-100ng/microl. The measurement is semiquantitative with a CV lower than 20%. A number of applications of this assay on a Labchip will be shown.     

HPLC法同时测定人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2

目的:建立高效液相色谱法同时测定人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2的方法.方法:采用ODSC18(4.6 mm×150 mm)色谱柱,流动相乙腈-0.05%磷酸水,梯度洗脱,流速1 Ml/min,检测波长203 nm,柱温35 ℃.结果:人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2分离效果良好,线性关...     

Simultaneous determination of cyclandelate and its metabolite in human plasma by capillary column gas-liquid chromatography

A method was developed for the simultaneous determination of cyclandelate and mandelic acid concentrations in plasma, involving extraction from plasma followed by trimethylsilylation and chromatography of the derivatives on a glass capillary column with hydrogen flame-ionization detection. Calibration graphs were linear down to at least 20 μg/ml for each substance. The precision was excellent with a pooled relative standard deviation of 6.3% and 6.4% for cyclandelate and mandelic acid serum samples, respectively. Concentrations below 500 ng/ml of each substance could be detected in human plasma. The method was developed for use in bioavailability and metabolism studies.     

Changes in vertical jump height, anthropometric characteristics, and biochemical parameters after contrast training in master athletes and physically active older people

The purpose of this study was to determine the effects of 16 weeks of contrast training (CT) on older adults (with different levels of physical conditioning) in vertical jump performance (squat jump [SJ], countermovement jump [CMJ], CMJ during 15 seconds [CMJ15], depth jump [DJ]), body weight, fat percentage, muscle mass (MM), muscle cross-sectional area ([CSA] of the arm and thigh) and biochemical parameters (creatine kinase [CK], creatinine, and urea). Sixteen older (63.55 ± 6.89 years) recreational master runners (A) and 16 physically active older people (60.30 ± 5.18 years) though not athletes (NA), participated in the CT using a combination of heavy-resistance and explosive exercise. The dependent variables were measured pretraining and posttraining. The CT resulted in significant improvements (α = 0.05) for both groups in jump performance. The SJ height improved in NA by 21.68% and in A by 21.81%, the CMJ height increased in NA by 21.51% and in A by 14.81%, the DJ height increased in NA by 26.45% and in A by 7.43%, and CMJ15 increased in NA by 6.20% and in A by 6.17%). Significant improvements in MM (16.44% in NA and 14.78% in A), thigh CSA (19.68% in NA and 21.67% in A), and arm CSA (7.43% in NA and 5.52% in A), and significant decreases in creatinine (8.65%) and CK (25.49%) in A were observed. In conclusion, CT improved vertical jump performance and MM in both groups, regardless of the training history and current physical activity of each group. These improvements were accompanied by a slight decrease in body fat but no changes in total body weight. These findings suggest that CT can have a significant effect on maximal jump height and MM in NA and A.     

Effects of straw mulching on soil temperature, evaporation and yield of winter wheat: field experiments on the North China Plain

Straw mulching is an effective measure to conserve soil moisture. However, the existence of straw on the soil surface also affects soil temperature, which in turn influences crop growth, especially of winter crops. Five‐year field experiments (2000–2005) investigated the effects of straw mulching and straw mass on soil temperature, soil evaporation, crop growth and development, yield and water use efficiency (WUE) of winter wheat (Triticum aestivum L.) at Luancheng Station on the North China Plain. Soil is a moderately well‐drained loamy soil with a deep profile at the station. Two quantities of mulch were used: 3000 kg ha^?1 [less mulching (LM)] and 6000 kg ha^?1 [more mulching (MM)], representing half and all of the straw from the previous crop (maize). In the control (CK), the full quantity of mulch was ploughed into the top 20 cm of soil. The results showed that the existence of straw on the soil surface reduced the maximum, but increased the minimum diurnal soil temperature. When soil temperature was decreasing (from November to early February the next year), soil temperature (0–10 cm) under straw mulching was on average 0.3°C higher for LM and 0.58°C higher for MM than that without mulching (CK). During the period when soil temperature increased (from February to early April, the recovery and jointing stages of winter wheat), average daily soil temperature of 0–10 cm was 0.42°C lower for LM and 0.65°C lower for MM than that of CK. With the increase in leaf area index, the effect of mulching on soil temperature gradually disappeared. The lower soil temperature under mulch in spring delayed the development of winter wheat up to 7 days, which on average reduced the final grain yield by 5% for LM and 7% for MM compared with CK over the five seasons. Mulch reduced soil evaporation by 21% under LM and 40% under MM compared with CK, based on daily measuring of microlysimeters. However, because yield was reduced, the overall WUE was not improved by mulch.     

Heterogeneity of Escherichia coli-expressed human muscle creatine kinase

Creatine kinase (CK) plays an important role in maintaining a constant ATP:ADP ratio during periods of high energy usage. Elevated levels of CK give an early indication of myocardial infarction. The enzyme has four major isozymes with heterogeneity being observed for each of them. In many cases the source of the heterogeneity is unclear. However, some of the isoforms are known to result from exposure to serum proteases, and analysis of the plasma isoforms provides an estimate of the time of onset of the infarction. Somewhat surprisingly, isoelectric focusing (IEF) experiments provided evidence of heterogeneity in human muscle CK (HMCK) expressed in E. coli. To investigate this further, HMCK was purified to apparent homogeneity utilizing Blue Sepharose affinity chromatography and HiPrep Q anion exchange chromatography. Additional purification on a PBE 94 chromatofocusing column resulted in four fractions, three of which, HMCK I - III, were characterized. The three isoforms are all active and have similar kinetic parameters. They exhibited identical bands on SDS PAGE but different anodal mobility on non-denaturing gels. Modification of C-terminal and/or cysteine residues has been ruled out, and deamidation of asparagine or glutamine residue(s) is proposed to be the cause of isoform formation. In addition each of these isoforms showed a similar four-band pattern on a carrier ampholytes-based IEF gel. Two-dimensional IEF analysis showed that an equilibrium was established between the four bands, suggesting that the four components were unstable and generated only when the protein was subjected to IEF.     

Enantioselective determination of alprenolol in human plasma by liquid chromatography with tandem mass spectrometry using cellobiohydrolase chiral stationary phases

A fast, sensitive, and enantioselective LC-MS/MS bioanalytical method was developed and validated for the direct determination of individual alprenolol enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP) along with supported liquid extraction (SLE) procedures. Complete baseline separation of enantiomeric alprenolol was achieved within 2 min in reversed phase chromatography at 0.9 ml/min. SLE in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.500-500 ng/ml for each alprenolol enantiomer using 0.0500 ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed < or = 7.3% relative standard deviation (RSD) and -6.2 to 8.0% relative error (RE) for both alprenolol enantiomers.     

Heterogeneous cellular expression of creatine kinase isoenzyme during normal rat heart development

The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.     

Temperature, media, and point of induction affect the N-terminal processing of interleukin-1beta

The expression of recombinant proteins in bacterial hosts may alter the biophysical properties of the protein of interest as a result of differences in post-translational processing from that observed when produced in the native cell. For example, recombinant human interleukin-1beta (IL-1beta) is produced as three isoforms when expressed in the Escherichia coli strain BL-21(DE3). These isoforms are produced by the non-homogeneous processing of the N-terminal methionine residue by the endogenous bacterial aminopeptidase and differ in the first residue (1-met, 1-ala, and 1-pro). Importantly, these isoforms have significantly different binding affinities for the IL receptor protein. Varying the temperature, media composition, and point of induction affects this N-terminal processing to favor one of the three isoforms of IL-1beta. We found changes in media composition and/or point of induction affected the abundance of the isoforms by as much as 15-fold. The 1-pro isoform decreased from 60.9 to 4.7% in Luria broth (LB) and minimal media (MM), respectively, when protein expression was induced at an OD600 of 0.9. Conversely, the abundance of the 1-met isoform is much higher in MM than in LB showing the reverse effect (2.6 and 50.7% in LB and MM, respectively, at an OD600 of 0.9), and the degree to which they are favored depends significantly upon the induction point. Our results show that it is possible to favor the expression of various N-terminal isoforms of IL-1beta by adjusting the environmental growth conditions. Given that the initiator methionine residue is necessary for expression in bacterial hosts and is known to affect the stability of other recombinant proteins our approach may be a useful general method for determining the optimal conditions for expressing and purifying pure, homogenous samples of recombinant proteins for structural and biological studies.     

Simultaneous Detection of Fenitrothion and Chlorpyrifos-Methyl with a Photonic Suspension Array

A technique was developed for simultaneous detection of fenitrothion (FNT) and chlorpyrifos-methyl (CLT) using a photonic suspension array based on silica colloidal crystal beads (SCCBs). The SCCBs were encoded with the characteristic reflection peak originating from the stop-band of colloidal crystal. This approach avoids the bleaching, fading or potential interference seen when encoding by fluorescence. SCCBs with a nanopatterned surface had increased biomolecule binding capacity and improved stability. Under optimal conditions, the proposed suspension array allowed simultaneous detection of the selected pesticides in the ranges of 0.25 to 1024 ng/mL and 0.40 to 735.37 ng/mL, with the limits of detection (LODs) of 0.25 and 0.40 ng/mL, respectively. The suspension array was specific and had no significant cross-reactivity with other chemicals. The mean recoveries in tests in which samples were spiked with target standards were 82.35% to 109.90% with a standard deviation within 9.93% for CLT and 81.64% to 108.10% with a standard deviation within 8.82% for FNT. The proposed method shows a potentially powerful capability for fast quantitative analysis of pesticide residues.     

The supramolecular organization and functions creatine kinase of system

The Creatine kinase (CK) SYSTEM represents key in a power exchange mediators the structure capable to plural interactions with the majority energy making (Glycolysis and mitochondriuns) and energy consuming (ATPases) structures at use of one multifunctional metabolits--creatine and providing transport macroergs inside a cell. Mitochondrions CK provides synthesis creatine phosphates (CP) from cytoplasmic creatine and energy mitochondriums ATP. CP energetically also is structurally more favourable than ATphi. The MM, MB and BB isoforms provide splitting Kphi and synthesis ATphi for M-ATPases, Ca-ATPases and Na-K-ATPases accordingly. Questions of regulation of activity of enzyme, both in ontogenesis, and in blood are discussed.     

Quantification of creative kinase isoenzymes by a radioimmunoassay

Summary It is not known whether loss of enzyme activity from the circulation is due to denaturation, inactivation or removal of intact enzyme molecules. This is in part due to the lack of an assay to measure enzyme protein concentration since available assays measure only enzyme activity. Radioimmunoassays for plasma enzymes and isoenzymes have not been possible because of oxidation in radioactive labelling by conventional methods and the problem of subunit dissociation. In the present study, antibodies specific to the B and M subunits of creatine kinase isoenzymes were obtained by immunization of rabbits with canine BB and MM creatine kinase. Anitgens (MM and BB) were radioactively labelled with ¹²⁵I by acylation, avoiding the problem of oxidation and subunit stabilized by mercaptoethanol (0.020 m) and Trisbuffer (1.6 m). A radioimmunoassay capable of detecting picogram amounts of CK isoenzymes was developed which measures the concentration of enzyme protein rather than activity. The method was shown to provide a sensitive quantitative method for analysis of plasma CK isoenzymes in dogs after myocardial infarction produced by coronary occlusion. This technique may provide a prototype for the development of radioimmunoassays for other plasma isoenzymes and should help to elucidate the nature of the disappearance of isoenzymes from the circulation.Work from the authors' laboratory was supported in part by the National Institutes of Health Grant HL 17646, SCOR in Ischemic Heart Disease     

Polyamines-an improved automated ion-exchange method

An accurate, precise, and improved automated cation-exchange chromatographic method with ninhydrin detection for the analysis of di- and polyamines (putrescine, cadaverine, spermidine, and spermine) has been developed. We have shown that different types of biological fluids such as urine, blood plasma, blood sera, tissue extracts, and cancer cell culture media can be analyzed under identical chromatographic conditions. The simplicity and precision of the method was achieved by eliminating the sample pre-separation and using an internal standard technique. Thus, not only has the sample preparation been simplified, but the accuracy and precision and sensitivity of the method have been greatly improved. Twenty-four unattended analyses were performed each day. With minor modifications of the instrument a two-fold analytical output can be achieved with analysis time cut to 30 min. The ruggedness and applicability of the method has been demonstrated in our laboratory during the past six months. More than two thousand urine and hundreds of other physiological samples have been analyzed by this method with a relative standard deviation from 3.3 to 7.8%, and recoveries of 94 to 97%.This automated ion-exchange chromatographic method for the polyamines will be useful to researchers in biological markers programs for monitoring the course of cancer and effectiveness of chemotherapy.     

Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks

We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%–99.9%), 100% (95% CI: 98.6%–100%) and 0.997 (95% CI: 0.987–1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R² = 0.84; p < 0.001). The agreement between results obtained with pairs of serum and milk samples was very high: there was matching classification in 96% (910/947) of paired samples (kappa = 0.92; p < 0.001). Individual milk samples may therefore be used, instead of serum samples, in the MM3-SERO ELISA, for reliable detection of seropositive cows. Testing bulk tank milk samples enabled detection of infected herds, even when the within-herd prevalence of infection was as low as 12%. We conclude that the MM3-SERO ELISA is a sensitive and highly specific test for serodiagnosis of bovine fasciolosis, and can be used with individual samples of either serum or milk. Use of the assay with bulk milk samples enables estimation of the within-herd prevalence of infection.     

An improved procedure for characterization of spatial and temporal evolution of immobilized cells in gel membranes

An improved procedure that allows the simple and reproducible characterization of spatial and temporal distribution of immobilized biomass in gel membranes was developed. This procedure involves three main steps in the preparation of membrane samples, the use of a standard microtome to obtain membrane slices, and the measurement of cell concentration by spectrophotometry. The key improvement in this procedure is to prepare the membrane samples by clamping them between two glass plates and storing them in a -80 degrees C freezer for a specified period of time depending on the membrane thickness. With this simple pre-treatment, the membrane samples were frozen in an ideal physical state to be cut into flat, consistent, slices using a commercial freezing sledge microtome, thus providing accurate and reproducible results. As a validation case study, a gel membrane bioreactor was constructed in which an alginate gel membrane with immobilized Lactobacillus rhamnosus cells was flanked by two well-mixed chambers with identical fermentation media. The improved procedure was employed to experimentally determine the intra-membrane cell distribution in the alginate membranes during fermentation. The experimental results showed a heterogeneous "U-shape" biomass distribution across the membrane, with the highest cell concentration at the membrane-solution interface. High reproducibility and accuracy were verified by a low average standard deviation (<5%) and a high biomass recovery ratio (>90%), respectively.     

Purification and some properties of two creatine kinase isoforms from herring (Clupea harengus) spermatozoa

Creatine kinase (CK, EC 2.7.3.2) isoforms play important role in energy homeostasis and together with easily diffusible compounds like creatine and phosphocreatine maintain a cellular energy buffer and intracellular energy transport system. The CK activity in spermatozoa is the highest from all studied tissues in herring. It was detected that the two CK isoforms, CK1 and CK2, are characteristic only for spermatozoa and are not expressed in other herring tissues. Isolation and purification procedures allowed obtaining purified enzymes with specific activity of the 345 micromol/min/mg for CK1 and 511 micromol/min/mg for CK2. Native Mr's of the CK1 and CK2 determined by gel permeation chromatography were about 330,000 and 90,000, respectively. These results indicate that CK1 form has octameric structure and CK2 is a dimer mostly characteristic for cytosolic CK enzymes. In immunoblotting studies with antisera against CK2, the response was observed for CK2 and there was no response for CK1 and two other isoforms from herring skeletal muscle. These findings make the herring isoforms an interesting model for studies on the fish CK biochemical properties.     

High throughput method for the determination of organochlorine pesticides and polychlorinated biphenyls in human serum

An improved method for the determination of selected organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum was developed. The method requires low volume of serum (500 microl) and 48-96 samples per day can be prepared by one analyst without special automatic equipment. Initial extraction was performed using 96-well solid-phase extraction disk plates and was followed by a clean-up with silica gel/sulfuric acid. Different denaturation, elution and clean-up conditions were tested. Quantification was carried out by gas chromatography equipped with electron capture detector (GC-ECD) or mass spectrometer (GC-MS). Recoveries of PCB congeners 28, 52, 101, 118, 138, 153 and 180 and OCPs HCB, beta-HCH, p,p'-DDE and p,p'-DDT at two spiking levels (n=8) varied from 57 to 120%, and intra-day relative standard deviation from 1 to 11%, both depending on spiking level and compound. Inter-day relative standard deviation was <15% in all cases. Limit of quantification (LOQ) for these PCBs ranged from 0.08 to 0.13 ng/ml and for these OCPs from 0.16 to 0.40 ng/ml. The optimized method was applied to the analysis of 1000 serum samples from different places of Spain.     

Development of a packed-column supercritical fluid chromatography/atmospheric pressure chemical-ionisation mass spectrometric technique for the analysis of atropine

A packed-column supercritical fluid chromatography/atmospheric pressure chemical-ionisation mass spectrometry (pSFC-APCI/MS) method has been developed for the determination of atropine from Atropa belladonna L extracts. The technique does not require any kind of derivatisation prior to the analysis. The optimum conditions were studied by using the pure substance in methanol (MeOH). All samples were simply dissolved in MeOH and injected into the mobile phase. Detection was achieved by using mass spectrometry (MS) with atmospheric pressure chemical ionisation (APCI). Terbutaline was used as an internal standard for the determination of the analytical reproducibility. The supercritical carbon dioxide (scCO(2)) mobile phase was modified by 15% MeOH containing 0.5% trifluoroacetic acid (TFAA) and 0.5% diethylamine (DEA) additives. Concentrations of atropine were determined with a relative standard deviation of less than 1% by the pSFC-APCI/MS procedure for a sample containing atropine and terbutaline. The correlation coefficient was 0.997 and detection limit 700 pg. The absolute retention time was 9.87 min with a standard deviation of 5.2x10(-3) min and a relative standard deviation of 0.61% with respect to terbutaline.     

Improved and simplified liquid chromatography/electrospray ionization mass spectrometry method for the analysis of underivatized glucosamine in human plasma

Glucosamine is an amino monosaccharide reagent. It is difficult to assay using typical reversed-phase column due to the early elution, by optimizing the chromatographic conditions, especially the analytical column and the mobile phase composition, an improved analytical method was developed and validated, which offers rapid, sensitive and specific determination of glucosamine in human plasma. Following protein precipitation, the analyte and internal standard (valibose) were separated using an isocratic mobile phase on an Inertsil CN-3 column and detected by mass spectrometry in the multiple reaction monitoring mode using the respective precursor to product ion combinations of m/z 180/72 for glucosamine and m/z 252/198 for valibose. The chromatographic time was just 4.2 min for each sample, which made it possible to analyze more than 120 human plasma samples per day. The method exhibited a linear dynamic range of 4.00-4000 ng/mL for glucosamine in human plasma. The lower limit of quantification (LLOQ) was 4.00 ng/mL with a relative standard deviation of less than 10.9%. Acceptable precision and accuracy were obtained for the plasma concentrations over the standard curve range. By monitoring the two different MRM transitions, it was proved that no endogenous glucosamine was found in human plasma. The validated method has been successfully used to analyze human plasma samples for application in a bioequivalence study.