Metabolic changes in human red cells during incubation of whole blood in vitro.
ABSTRACT: 1. Whole blood was incubated at 37 degrees , while being dialysed against a large volume of iso-osmotic bicarbonate buffer, pH7.4. The buffer contained glucose and the essential inorganic components of blood plasma in proportion. 2. After 3hr. of incubation in vitro there is a loss of red-cell 2,3-diphosphoglycerate. 3. Isotope experiments show that this is due to an accelerated rate of destruction of this compound. 4. Simultaneously, there is an increase in the median of red-cell osmotic fragility. 5. After extended periods of incubation there is a decrease in the metabolic rate and a decrease in the ratio of the rates of lactate production to glucose consumption. 6. There is a continuous loss of total adenine nucleotide and, after the first 12hr. of incubation, a tendency for the intracellular Na(+) and K(+) to equilibrate with the plasma. 7. The standard deviation of red-cell osmotic fragility expressed among the red-cell population increases exponentially with the time of incubation.
Project description:1. Spermine and spermidine were the main polyamines detectable in Bacillus stearothermophilus. 2. When grown at 65 degrees B. stearothermophilus contained lower concentrations of polyamines per mg. of RNA than when grown at 45 degrees or at 55 degrees . 3. Ribosomes isolated from B. stearothermophilus in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride contained sufficient polyamines to neutralize between 4% and 9% of their RNA phosphorus. 4. Removal of polyamines from the ribosomes by dialysis against m-potassium chloride did not appreciably alter the hypochromicity or thermal denaturation profiles of the ribosomes when measured in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride, though it did cause a loss of ribosome particles sedimenting at greater than 78s. 5. When ribosomes were dialysed against acridine orange solutions acridine orange bound to the ribosomes and did not displace spermine, but when a mixture of ribosomal RNA and spermine was dialysed against acridine orange the acridine orange displaced the spermine. It is concluded that polyamines in the ribosomes are less accessible for displacement by acridine orange than when polyamines are bound to ribosomal RNA.
Project description:A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide-0.33m-potassium chloride-10mm-tris-hydrochloric acid buffer, pH7.4, at 33 degrees for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.
Project description:1. The effects of sucrose and KCl on the loss of latency of lysosomal enzymes caused by incubation at 37 degrees C, pH 7.4, were examined by using Triton-filled lysosomes from rat liver and two fractions from livers of rats not injected with Triton. 2. After incubation, the percentage free activity of lysosomal enzymes was measured before and after cooling to 0 degrees C in order to determine the amount of latency lost at 37 degrees C without cooling and the additional amount lost on cooling the incubated lysosomes to 0 degrees C. 3. The latency that is lost without cooling is first decreased and then increased by increasing the osmotic strength of the incubation medium with KCl, or with sucrose in the presence of KCl. However, if the osmotic strength is increased with sucrose alone, loss of latency is decreased up to 0.25M-sucrose, but is increased only slightly at higher sucrose concentrations. Apparently the lysosome is permeated by hyperosmolar KCl but not by sucrose during incubation. 4. If the osmotic strength of the assay medium is increased with KCl, the loss of latency caused by incubation for 60 min in hyperosmolar KCl is repressed. Thus it appears that a KCl-permeated lysosome can be obtained which is relatively stable until exposure to lower osmolarities. 5. The loss of latency caused by cooling incubated lysosomes to 0 degrees C is largely eliminated if the osmotic strength of the medium in which the lysosomes are cooled is raised sufficiently with either sucrose or KCl. 6. Osmotic-fragility curves were obtained after incubation for 1 and 60 min at iso-osmoticity (0.2M-KCl or 0.25 M-sucrose). Although little loss of latency occurs at iso-osmoticity, lysosomes incubated for 60 min display greatly increased fragility on exposure to hypo-osmolar KCl, hypo-osmolar sucrose or hyperosmolar KCl. 7. It is suggested that permeability to KCl at 37 degrees C and the increase in fragility on exposure to hypo-osmolar conditions are both consequences of injury, probably from enzymic action, sustained by the lysosomal membrane during incubation at 37 degrees C.
Project description:1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.
Project description:Garden snails (<i>Cornu aspersum</i>) have been sacrificed by drowning the snails overnight in water. The visceral organs (inside the shell organs) have been separated from the foot as well as the shell and homogenized using tris-HCl buffer, pH 5. The homogenate of visceral organs was dialysed in distilled water at 4?°C for 18?h where after the dialysed material was used to bio-convert the cellulose component of various waste paper materials into fermentable sugars such as glucose. Saccharification of the waste cellulose materials was performed with the extracted snail cellulase during ten consecutive incubation periods of 2?h each. The amount of sugars produced during cellulase action on waste cellulose was determined by the dinitrosalicylic acid (DNS) method. All incubations were performed in triplicate and the percent saccharification of each paper material was determined as a fraction of the paper material exposed to cellulase action. •Cellulase extracted from brown garden snail•Saccharification of waste paper using garden snail cellulase.
Project description:The present study aimed to characterize the function and hemorheology of red blood cells (RBCs) recovered during liver transplantation surgery in patients with hepatitis B and decompensation. A total of 15 hepatitis B patients with decompensation who underwent liver transplantation surgery were included in the present study. Blood samples were recovered during the liver transplantation surgery using an Autologous Blood Recovery System. The morphology and structure of RBCs were characterized and compared between pre-operative and recovered blood samples. In addition, the physiological functions of RBCs were measured and compared between pre-operative and recovered blood samples. No significant differences in the morphological score, 2,3-diphosphoglycerate, Na+K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, malondialdehyde and osmotic fragility were identified between RBCs in the pre-operative and recovered blood samples. The level of free hemoglobin in RBCs of the recovered blood samples was significantly higher than in the pre-operative blood samples (P<0.05). Medium- and high-shear blood viscosities in the recovered blood samples were significantly lower than those observed in the pre-operative blood samples (P<0.05). Casson viscosity in the recovered blood samples was significantly higher compared with the pre-operative blood samples. However, no significant differences (P>0.05) in the low-shear blood viscosity, plasma viscosity, relative blood viscosity, erythrocyte aggregation index or Casson yield stress were identified between recovered and pre-operative blood samples. These findings suggested that autologous blood transfusion in liver transplantation surgery in patients with hepatitis B and decompensation had no significant influence on the morphology, structure, function and hemorheology of RBCs.
Project description:The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction.
Project description:When rat liver homogenate or its postmitochondrial supernatant was incubated with L-cysteine, but not D-cysteine, ornithine decarboxylase (ODC) lost more than half of its catalytic activity within 30 min and, at a slower rate, its immunoreactivity. The inactivation correlated with production of H2S during the incubation. These changes did not occur in liver homogenates from vitamin B6-deficient rats. A heat-stable inactivating factor was found in both dialysed cytosol and washed microsomes obtained from the postmitochondrial supernatant incubated with cysteine. The microsomal inactivating factor was solubilized into Tris/HCl buffer, pH 7.4, containing dithiothreitol. Its absorption spectrum in the visible region resembled that of Fe2+ X dithiothreitol in Tris/HCl buffer. On the other hand FeSO4 inactivated partially purified ODC in a similar manner to the present inactivating factor. During the incubation of postmitochondrial supernatant with cysteine, there was a marked increase in the contents of Fe2+ loosely bound to cytosolic and microsomal macromolecules. Furthermore, the content of such reactive iron in the inactivating factor preparations was enough to account for their inactivating activity. These data suggested that H2S produced from cysteine by some vitamin B6-dependent enzyme(s) converted cytosolic and microsomal iron into a reactive loosely bound form that inactivated ODC.
Project description:Hereditary spherocytosis (HS) is a clinically heterogeneous disease characterized by mild to moderate hemolysis resulting from red cell membrane protein defects. Diagnostic tests include hemogram, reticulocyte count and blood smear evaluation, osmotic fragility, cryohemolysis, SDS-PAGE, flow cytometry using eosin-5'-maleimide (EMA) and genetic studies. We evaluated the flow cytometric EMA-binding test and compared it with osmotic fragility in 51 consecutive cases of suspected HS aged between 10 days and 62 years. In addition, 4 cases suspected on blood smears underwent EMA testing alone. The 16 EMA-positive cases who were determined to have HS had overlapping hemoglobin levels and reticulocyte counts with the 35 patients with normal EMA results, highlighting the importance of the flow cytometric test in providing a definitive diagnosis. Flow cytometric EMA binding test was thus a simple and relatively faster method to confirm HS in our experience.
Project description:Extracorporeal circulation is accompanied by changes in red blood cell morphology and structural integrity that affect cell function and survival, and thereby may contribute to the various side effects of heart-lung machine-assisted surgery. Our main objectives were to determine the effect of circulation of red blood cells in a stand-alone extracorporeal circuit on several parameters that are known to be affected by, as well as contribute to red blood cell aging. As a source of RBCs, we employed blood bank storage units of different ages. In order to assess the relevance of our in vitro observations for the characterization of extracorporal circulation technology, we compared these changes in those of patients undergoing extracorporeal circulation-assisted cardiac surgery. Our results show that circulation in a heart-lung machine is accompanied by changes in red blood cell volume, an increase in osmotic fragility, changes in deformability and aggregation behavior, and alterations in the exposure of phosphatidylserine and in microvesicle generation. RBCs from 1-week-old concentrates showed the highest similarities with the in vivo situation. These changes in key characteristics of the red blood cell aging process likely increase the susceptibility of red blood cells to the various mechanical, osmotic, and immunological stress conditions encountered during and after surgery in the patient's circulation, and thereby contribute to the side effects of surgery. Thus, aging-related parameters in red blood cell structure and function provide a foundation for the validation and improvement of extracorporeal circulation technology.