Western blotting assay of transketolase concentration in human hemolysates

Using a rabbit anti-human transketolase antiserum and Western blotting we can determine nanogram amounts of transketolase in human hemolysates quantitatively. Transketolase concentration in 18 apparently healthy subjects was 55.7 +/- 12.1 micrograms/g Hb (mean +/- SD). Transketolase concentration correlated positively with the enzyme activity both with and without in vitro addition of thiamin pyrophosphate. However, the former had a closer correlation (r = 0.8418, P less than 0.001) than the latter (r = 0.6703, P less than 0.01). A heavy drinker with an extremely low transketolase activity had proportionally low concentration to the activity. These results indicate that transketolase in hemolysates, whether it is holoenzyme or apoenzyme activated in vitro, has an identical specific activity among all subjects studied and that the reduced activity of transketolase in alcoholics is due to the reduced content of the enzyme protein. This method is applicable to study the dynamics and the abnormality of apotransketolase in human hemolysates.     

A high-throughput assay of NK cell activity in whole blood and its clinical application

Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as ⁵¹Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.     

A colorimetric assay to quantify dehydrogenase activity in crude cell lysates

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7 degrees C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.     

建立转酮酶酶活测定方法及应用

摘要：目的 转酮酶是非氧化磷酸化戊糖途径中的关键酶。5-磷酸木酮糖是分析测定转酮酶酶活性的底物,然而因该底物难以化学合成,生产成本高,从而导致厂家停止生产,目前市场上无法得到。因此有必要建立起新的转酮酶酶活测定方法。方法 （1）将酿酒酵母的木酮糖激酶基因（XKS1）克隆于pET30a载体。（2）纯化木酮糖激酶。（3）建立酶偶联反应,以木酮糖为底物,将其转化为5-磷酸木酮糖,用于转酮酶酶活的测定。结果 （1）成功地将XKS1基因克隆于pET 30a得到质粒pXZ-X004。（2）质粒pXZ-X004诱导表达产生C端His-tag标签的XK,用Ni柱分离得到纯化的木酮糖激酶(XK)。（3）纯化的XK活性为21.0 U/mg protein,当用12.5%的甘油保存于?80 ℃环境1个月,仍有74%的活性。（4）用纯化的XK建立了转酮酶（TK）酶活测定新方法,当TK酶活低至0.00875 U时仍能够利用新方法检测。结论 本实验通过克隆和表达XKS1基因,成功地建立了TK酶活性测定的新方法,并应用于木糖代谢工程菌株TK酶活的测定,为医学和生态学领域中TK酶活的分析奠定了基础。     

Cytochemical method for detecting dehydrogenase activity in the leukocytes of native blood smears

A cytochemical method of detection of dehydrogenases in blood leucocytes is proposed. Native smears are dried up in the air to be incubated at 37 degrees C in gel-containing medium composed of polyvinyl alcohol, sucrose, a corresponding substrate, cofactors and inhibitors of cytochrome oxidases activity. Using corresponding media, activities of succinate, malate, glutamate, lactate-, alpha-glycerophosphate, alcohol, beta-oxybutyrate and glucoso-5-phosphate dehydrogenases were revealed. Half-reduced diformazan providing diffuse rosy staining of cells was removed after the incubation, and the incubation medium was washed out by rinsing the smears in 60% acetone solution. As a result monochromatic micropreparations may be received. Finally, smears are fixed in formalin. The above method provides a reduced loss of enzymes, preserves a good cell morphology and eliminates non-dehydrogenase effects of tetrazolium reduction into formazan.     

建立转酮酶酶活测定方法及应用

目的转酮酶是非氧化磷酸化戊糖途径中的关键酶。5-磷酸木酮糖是分析测定转酮酶酶活性的底物,然而因该底物难以化学合成,生产成本高,从而导致厂家停止生产,目前市场上无法得到。因此有必要建立起新的转酮酶酶活测定方法。方法(1)将酿酒酵母的木酮糖激酶基因(XKS1)克隆于pET30a载体。(2)纯化木酮糖激酶。(3)建立酶偶联反应,以木酮糖为底物,将其转化为5-磷酸木酮糖,用于转酮酶酶活的测定。结果 (1)成功地将XKS1基因克隆于pET 30a得到质粒pXZ-X004。(2)质粒pXZ-X004诱导表达产生C端His-tag标签的XK,用Ni柱分离得到纯化的木酮糖激酶(XK)。(3)纯化的XK活性为21.0U/mg protein,当用12.5%的甘油保存于-80℃环境1个月,仍有74%的活性。(4)用纯化的XK建立了转酮酶(TK)酶活测定新方法,当TK酶活低至0.008 75U时仍能够利用新方法检测。结论本实验通过克隆和表达XKS1基因,成功地建立了TK酶活性测定的新方法,并应用于木糖代谢工程菌株TK酶活的测定,为医学和生态学领域中TK酶活的分析奠定了基础。     

Effects of cold acclimation on the activity levels of creatine kinase, lactate dehydrogenase and lactate dehydrogenase isoenzymes in various tissues of the rat.

The effects of cold acclimation on the activity levels of creatine kinase, lactate dehydrogenase and lactate dehydrogenase isoenzymes in various tissues/ organs of the rat (Rattus norvegicus) were investigated. Male Sprague-Dawley rats were divided into two groups. One group was housed at 4+/-1 degrees C (experimental group) and the other at 24+/-1 degrees C (control group) for six months. The rats were housed in single cages and had access to food and water ad libitum. The tissues/organs investigated were heart, liver, lung, kidney, gastrocnemius muscle and interscapular brown adipose tissue as well as serum. With the exception of lung, (which showed a decrease of 24%) total creatine kinase activity levels were significantly increased (P< 0.05) in all the tissues/organs investigated (17-51%) as well as serum (34%), in cold acclimated animals. Cold acclimation also resulted in significantly increased (P< 0.05) activity levels of lactate dehydrogenase in all the tissues/organs investigated (14-24%) as well as serum (35%). Cold exposure resulted in an increase of the activity levels of all the detectable isoenzymes of lactate dehydrogenase, although not always significant, in all the tissues/organs investigated as well as serum. The M(4)tetramer of lactate dehydrogenase was the only detectable isoenzyme in serum.     

Faster lactate transport across red blood cell membrane in sickle cell trait carriers.

The physical and physiological behavior of sickle cell trait carriers (AS) is somewhat equivocal under strenuous conditions, although this genetic abnormality is generally considered to be a benign disorder. The occurrence of incidents and severe injuries in AS during exercise might be explained, in part, by the lactic acidosis due to a greater lactate influx into AS red blood cells (RBCs). In the present study, the RBC lactate transport activity via the different pathways was compared between AS and individuals with normal hemoglobin (AA). Sixteen Caribbean students, nine AS and seven AA, performed a progressive and maximal exercise test to determine maximal oxygen consumption. Blood samples were obtained at rest to assess haematological parameters and RBC lactate transport activity. Lactate influxes [total lactate influx and monocarboxylate transporter (MCT-1)-mediated lactate influx] into erythrocytes were measured at four external [14C]-labeled lactate concentrations (1.6, 8.1, 41, and 81.1 mM). The two groups had similar maximal oxygen consumption. Total lactate influx and lactate influx via the MCT-1 pathway were significantly higher in AS compared with AA at 1.6, 41, and 81.1 mM. The maximal lactate transport capacity for MCT-1 was higher in AS than in AA. Although AS and AA had the same maximal aerobic physical fitness, the RBCs from the sickle cell trait carriers took up more lactate at low and high concentrations than the RBCs from AA individuals. The higher MCT-1 maximal lactate transport capacity found in AS suggests greater content or greater activity of MCT-1 in AS RBC membranes.     

Plasma and intracellular levels of lactate dehydrogenase, phosphohexose isomerase and lysozyme activity in acute leukemia

Plasma and intracellular levels of lactate dehydrogenase (LDH), phosphohexose isomerase (PHI) and lysozyme activities were investigated in 20 patients with acute myelocytic leukemia (AML), 18 patients with acute lymphatic leukemia (ALL) and 10 patients with chronic myelocytic leukemia in blast transformation (CML/BT). Though the plasma levels of LDH and PHI in all patients with acute leukemia were elevated as compared to control persons there was no distinctive pattern which could be of use in the classification of acute leukemia. On the other hand the intracellular levels of these enzymes could be of value in classifying acute leukemia. The leukemic lymphoblasts were characterized by low levels of PHI and lysozyme as compared to leukemic myeloblasts or to normal lymphocytes (p less than 0.01). The LDH/PHI ratio is also significantly higher in leukemic lymphoblasts than in leukemic myeloblasts or in normal lymphocytes (p always less than 0.01). These characteristics might also be made use of in identifying the blasts of CML/BT als "lymphoid" or "myeloid" in corresponding cases.     

A sensitive spectrophotometric assay of pyruvate dehydrogenase activity

The recently reported highly sensitive method for assay of acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) [H. H. Andres, A. J. Klein, S. M. Szabo, and W. W. Weber (1985) 145, 367-375] has been adapted for determination of pyruvate dehydrogenase activity. This method provides an improvement in sensitivity over extant spectrophotometric methods and circumvents limitations of assays using radioactive pyruvate. In addition, the assay is simple and inexpensive and can be readily adapted for measurement of enzyme activity in crude tissue extracts or homogenates.     

Interaction of liposomes with human leukocytes in whole blood

The uptake of multilamellar liposomes into human leukocytes in whole blood in vitro was evaluated on the basis of the cellular association of liposomal markers (3H-labelled cholesterol, lipid phase; [14C]inulin, aqueous phase). The entry of liposomes into human blood leukocytes was linear for 60 min and was mediated by a saturable mechanism displaying affinity constants of 0.28 +/- 0.17 and 0.16 +/- 0.05 mM liposomal lipid (means +/- S.E.) for liposomal lipid and aqueous phase markers, respectively. Amicon filtration analysis of incubation mixtures containing blood and liposomes (phosphatidylcholine:dicetyl phosphate:cholesterol, 70:20:10) showed that 34% of [14C]inulin was lost (neither liposome-associated nor cell-associated) after 60 min. By preincorporating sphingomyelin (35 mol%) into multilamellar liposomes, the leakage of the model aqueous phase marker inulin was reduced to 8% after 60 min, thus enhancing the drug carrier potential of liposomes in blood. As a consequence of their interaction with liposomes, the polymorphonuclear leukocytes in whole blood decreased in apparent buoyant density, while maintaining their viability. These results indicate that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up multilamellar liposomes.     

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates

Fluorescence emission of free protoporphyrin IX (PPIX, em. approximately 626 nm), zinc protoporphyrin IX (ZPP, em. approximately 594 nm) and fluorescent heme degradation product (FHDP, em. approximately 466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F(365/594)) correlates with the ZPP fluorescence obtained from acetone/water extraction (R(2) = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R(2) = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.     

Alterations in lactate dehydrogenase isoenzymes and phosphofructokinase activity in the hormonally induced G-XII cell line

G-XII cell line was earlier developed in our laboratory from the granulosa lutein cells of goat ovaries, subjected to sustained stimulation by ovine luteinizing hormone. It is a non-clonogenic and non-tumorigenic cell line which secretes progesterone. In the present paper, we report some biochemical findings on the cell line studied between passages 10 and 15. The cells exhibited a shift in the lactate dehydrogenase isoenzyme pattern from the heart form to muscle form and a 300% increase in the phosphofructokinase activity compared to fresh granulosa cells.     

Viscoelastic properties of whole blood. Influence of fast sedimenting red blood cell aggregates

Red blood cell (RBC) aggregation is known to be of deciding influence on erythrocyte sedimentation-rate (ESR) and on whole blood viscoelastic properties. The rheological behaviour of blood collected from a control-group with normal ESR is compared to the viscoelastic behaviour of blood collected from two groups with high to very high ESR, whose individuals are suffering from chronical polyarthritis and Morbus Bechterew, respectively. The rheological properties are evaluated by means of an oscillating-flow capillary-rheometer where the viscous (eta') and elastic (eta") component of the complex viscosity (eta) is measured at a constant frequency of 2 Hz. Correcting for the varying hematocrit of the different blood samples according to an exponential equation, the viscoelastic data are found to be elevated in the groups with high ESR. For the viscous properties this is only due to the increase of the plasma viscosity. A correction for the plasma viscosity, however, shows that the viscous properties at low shear- rates (2s-1) are significantly reduced, whereas elastic properties in a range of medium shear-rates (10s-1 to 50s-1) are significantly increased (P less than 0.001, t-test of Student). This result is discussed to be due to the high packing density of the RBC in fast sedimenting aggregates. High packing density reduces the effective volume of the RBC but increases the stiffness of the aggregates.