Pyrimidine nucleotidases/phosphotransferases from human erythrocyte.

Two cytoplasmic pyrimidine 5'-nucleotidase have been purified from human erythrocytes to homogeneity and partially characterized. The two enzymes, indicated as PN-I and PN-II, preferentially hydrolyse pyrimidine 5'-monophosphates and 3'-monophosphates, respectively. The kinetic analysis demonstrate that pyrimidine 5'-nucleotidases, in the presence of suitable nucleoside substrates, can operate as phosphotransferases by transferring phosphate to various nucleoside acceptors, including nucleoside analogues known as important drugs widely used in chemotherapy.     

Acceptor Specificity Observed with Crude Preparation of Nucleoside Phosphotransferases

The acceptor specificities of bacterial nucleoside phosphotransferase were further investigated by phosphorylating various kinds of nucleoside analogues. The bacteria belonging to A group(5′-nucleotide former) specifically phosphorylated the primary alcohol at 5′-position of nucleosides and their analogues, such as adenine xyloside, psicofuranine and pseudouridine, while the others belonging to B group (3′(2′)-nucleotide former) the secondary alcohol at 3′(2′)-position. The phosphorylation at 5′-primary alcohol with the bacteria belonging to A group, however, was prohibited mainly by phosphoryl-or amino-radical at 3′-position, as observed in the case of 3′-nucleotide or amino-nucleoside (or puromycin), depending on the steric conformation around the 3′-position of acceptor. Besides, both types of nucleoside phosphotransferases were also able to phosphorylate nucleoside having a C-C-linkage between base and sugar moieties.     

Major Human Erythrocyte Glycoprotein spans the Cell Membrane

The polypeptide part of the principal glycoprotein on the surface of human erythrocytes extends through the membrane barrier to the interior surface of the cell membrane.     

Investigation of the Accompaniment of Calcium During Active Calcium Transport from Human Erythrocyte Ghosts

To determine whether a cell metabolite was involved in active calcium transport, the cell contents of human erythrocytes were subjected to high dilutions and the resultant ghosts were checked for their ability to actively transport calcium. It was found that the diluted erythrocyte ghosts did retain their capacity to actively transport calcium and that the characteristics of this transport process appeared to be unaltered by the high dilutions. Calcium analysis of the cell membrane and cell supernatant indicated that almost all of the calcium was lost from the cell solution rather than the cell membrane as active calcium transport proceeded. Therefore it appeared that calcium was able to cross the cell membrane without the aid of a cell metabolite. Investigations with layered erythrocytes indicated that the active transport of calcium was not assisted by centrifugation. Neither inorganic phosphate, pyrophosphate, nor an adenine nucleotide appeared to accompany calcium across the membrane as indicated by total phosphate and inorganic phosphate analysis and 260-nm readings of the deproteinized supernatant.     

Pyrimidine homonucleoside analogues from 2,5-anhydroaldose derivatives

3,4-Di-O-acetyl-2,5-anhydro-D-xylose diisobutyl dithioacetal (1) reacts with bromine to give a monobromo derivative which, on condensation with 2,4-diethoxy-pyrimidine or its 5-methyl analogue, affords the protected nucleoside derivatives 4 and 11, respectively; ammonolysis of 4 gave the cytosine “homonucleoside” 7, and hydrolysis of 11 gave the thymine “homonucleoside” 12. The same type of “homonucleoside” may be produced by cyclization of the sugar chain in a suitable acyclic-sugar nucleoside, as in the conversion of 1-S-ethyl-1-thio-1-(uracil-1-yl)-D-xylitol (16, obtained from tetra-O-acetyl-D-xylose diethyl dithioacetal, 9), by the action of one molar equivalent of p-toluenesulfonyl chloride, into a homonucleoside isolated as its diacetate 17; acyclic-sugar derivatives not susceptible to such cyclization afford instead the 5-p-toluenesulfonates, as exemplified by the conversion of the D-arabino analogue (13) of 16 into the 5′-ester 14. When cyclohexene is used to remove the excess of bromine in the preparation of nucleoside analogues from dithioacetals, the alkylsulfenyl bromide produced may react, by way of its cyclohexene adduct, with the hoterocyclic base to give cyclohexane-base adducts, for example, compounds 6 and 10.     

Yield Strength of Human Erythrocyte Membranes to Impulsive Stretching

Deformability while remaining viable is an important mechanical property of cells. Red blood cells (RBCs) deform considerably while flowing through small capillaries. The RBC membrane can withstand a finite strain, beyond which it ruptures. The classical yield areal strain of 2–4% for RBCs is generally accepted for a quasi-static strain. It has been noted previously that this threshold strain may be much larger with shorter exposure duration. Here we employ an impulse-like forcing to quantify this yield strain of RBC membranes. In the experiments, RBCs are stretched within tens of microseconds by a strong shear flow generated from a laser-induced cavitation bubble. The deformation of the cells in the strongly confined geometry is captured with a high-speed camera and viability is successively monitored with fluorescence microscopy. We find that the probability of cell survival is strongly dependent on the maximum strain. Above a critical areal strain of ∼40%, permanent membrane damage is observed for 50% of the cells. Interestingly, many of the cells do not rupture immediately and exhibit ghosting, but slowly obtain a round shape before they burst. This observation is explained with structural membrane damage leading to subnanometer-sized pores. The cells finally lyse from the colloidal osmotic pressure imbalance.     

Active Calcium and Strontium Transport in Human Erythrocyte Ghosts

Both calcium and strontium could be transported actively from erythrocytes if adenosine triphosphate, guanosine triphosphate, or inosine triphosphate were included in the hypotonic medium used to infuse calcium or strontium into the cells. Acetyl phosphate and pyrophosphate were not energy sources for the transport of either ion. Neither calcium nor strontium transport was accompanied by magnesium exchange, and the addition of Mg⁺⁺ to the reaction medium in a final concentration of 3.0 mmoles/liter did not promote the transport of either ion. In the absence of nucleotide triphosphates, the addition of 1.5 mmoles/liter of Sr⁺⁺ to the reaction solution did not bring about active calcium transport and similarly 1.5 mmoles/liter of Ca⁺⁺ did not bring about active strontium transport. The inclusion of 1.5 mmoles/liter of Ca⁺⁺ or Sr⁺⁺ in the reaction medium did not interfere with the transport of the other ion when the erythrocytes were infused with adenosine triphosphate.     

Yield Strength of Human Erythrocyte Membranes to Impulsive Stretching

Deformability while remaining viable is an important mechanical property of cells. Red blood cells (RBCs) deform considerably while flowing through small capillaries. The RBC membrane can withstand a finite strain, beyond which it ruptures. The classical yield areal strain of 2–4% for RBCs is generally accepted for a quasi-static strain. It has been noted previously that this threshold strain may be much larger with shorter exposure duration. Here we employ an impulse-like forcing to quantify this yield strain of RBC membranes. In the experiments, RBCs are stretched within tens of microseconds by a strong shear flow generated from a laser-induced cavitation bubble. The deformation of the cells in the strongly confined geometry is captured with a high-speed camera and viability is successively monitored with fluorescence microscopy. We find that the probability of cell survival is strongly dependent on the maximum strain. Above a critical areal strain of ∼40%, permanent membrane damage is observed for 50% of the cells. Interestingly, many of the cells do not rupture immediately and exhibit ghosting, but slowly obtain a round shape before they burst. This observation is explained with structural membrane damage leading to subnanometer-sized pores. The cells finally lyse from the colloidal osmotic pressure imbalance.     

Dynamics of Interaction of Phosphatidylcholine/Octadecylamine Liposomes with Human Erythrocyte Membranes: Electron Microscopic Study

Abstract

The interactions of octadecylamine and of positively charged liposomes (egg phosphatidylcholine/octadecylamine in molar ratio from 7:5 to 7:0.5) with human erythrocyte membrane have been studied by freeze-fracture and thin-section electron microscopy.

For the first time a very fast adsorption of liposomes to the cell membranes (less than 1 sec) is shown, and their intensive incorporation into plasma membrane (probably within the first 2-5 sec) without visible changes in cell morphology.

The prolonged incubation of the cells with liposomes results in certain morphologic changes: the transition of diskocytes to stomatocytes (30-80 sec) accompanied by the formation of isolated membrane vesicles in cell matrix (80-120 sec); the formation of pentalaminar contacts between plasma membrane of spherocytes and the membrane of isolated matrix vesicles (2.5-3 min); and the incorporation of matrix vesicle membranes into the spherocyte membranes (5 min+).

Possible molecular mechanisms underlying the observed structural changes are discussed briefly.     

The Surface of the Washed Human Erythrocyte as a Polyanion

The electrokinetic behaviour of normal erythrocytes is compared with that of trypsin-, N-bromosuccinimide-, and tosyl-treated erythrocytes. Reduction in the net negative charge with reduction in ionic strength of the suspending medium and also on treatment with N-bromosuccinimide and trypsin is discussed using a porous non-rigid polyanion as a model for the periphery of the cell membrane. It is deduced from the equivalent binding of chloride and thiocyanate ions and the absence of any effect on treatment of red cells with tosyl chloride, that normal, N-bromosuccinimide- and trypsin-treated cells are polyanionic in character. Reduction in erythrocyte charge on treatment with N-bromosuccinimide or trypsin is probably not due to the removal of phosphate groups from the interface, nor to physical adsorption of N-bromosuccinimide or trypsin. The charge reduction is probably produced by bond fission with possibly a net disappearance of carboxyl groups from the electrophoretic plane of shear either by loss from, or reorientation of, the membrane. The loss or reorientation of material associated with these carboxyl groups does not lead to any basic change in the character of the surface of the cell, nor to any obvious structural instability. The biconcave discoid form is maintained and there is no significant hemolysis of the erythrocytes even after contact with a solution of trypsin for 24 hours.     

Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte

The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.     

Pyrimidine Nucleotide-Stimulated Thromboxane A2 Release from Cultured GLIA

1. Uridine triphosphate (UTP), uridine diphosphate (UDP), cytidine triphosphate (CTP), and deoxythymidine triphosphate (TTP) caused concentration-dependent increases in the release of thromboxane A₂ (TXA₂) from cultured glia prepared from the newborn rat cerebral cortex. Although each of the pyrimidine nucleotides displayed similar potencies, CTP and TTP were considerably less effective than either UTP or UDP. The purine nucleotide ATP was equally as potent as the pyrimidine nucleotides but was marginally less effective than either UTP or UDP.2. The ability of UTP, UDP, TTP, and CTP to promote TXA₂ release from cultured glia was inhibited in a concentration-dependent manner by suramin and was markedly reduced when incubations were performed either in Ca²⁺-free medium or on cultures which had been maintained in serum-free growth medium for 4 days prior to experimentation.3. Challenges with UTP and UDP in combination were found to elicit a response which was no different from the effects of these nucleotides alone; in addition, their effects were reversed by the phospholipase A₂ inhibitor ONO-RS-082. A slight reduction in UTP-and UDP-stimulated TXA₂ release was observed in cultures grown in the presence of leucine methyl ester, a treatment reported to limit microglial survival.4. These results suggest that glia are targets for extracellular pyrimidine nucleotides and that their ability to release eicosanoids from these cells may be important in the brain's response to damage.     

The Polymeric State of Actin in the Human Erythrocyte Cytoskeleton

Reports on the polymeric state of actin in the red cell have been diverse. We have used phalloidin to stabilize the actin in erythrocyte ghosts prior to extraction in low ionic strength media. A mild proteolytic digestion and Sepharose 4B gel filtration enable an F-actin polymer to be isolated in pure form [1]. Detailed size analysis of this polymer in a range of experiments suggests that actin exists in the erythrocyte principally as a polymer of 100 nm length composed of 30 monomers in a double helical chain 15 monomers long with an estimated molecular weight of 1.3 × 10⁶ daltons.     

人铜锌超氧化物歧化酶的大规模生产工艺

本文以干扰素生产中废弃的人红细胞为原料,采用乙醇－氯份沉淀→磷酸氢二钾盐析→丙酮沉淀手续和ＤＥＡＥ—５２层析步骤,进行了人铜锌超氧化物歧化酶（ＣｕＺｎ－ＳＯＤ）大规模生产工艺的实验研究。结果表明,生产的酶制剂中含量为９０．６％,比活性在２０００ＭｃＣｏｒｄ—Ｆｒｉｄｏｖｉｃｈ单位／ｍｇ蛋白以上,无菌、无热原质、安全性等项指标符合国家卫生部对血液制品的要求。     

Hydrated Water Molecules of Pyrimidine/Purine/Pyrimidine DNA Triple Helices as Revealed by FT-IR Spectroscopy: A Role of Cytosine Methylation

Abstract

Hydrated water molecules of pyrimidine/purine/pyrimidine DNA hairpin triplex was studied by a comparison of triplex (CC·AG6) formed by a host oligodeoxypyrimidine of 5′- d(TC)₃T₄(CT)₃ (CC) with a target hexadeoxypurine 5′-d(AG)₃ (AG6) strand and by triplexes (MM·AG6, MC·AG6, and CM·AG6) formed by oligonucleotides with the exact sequences as above except 5-methylcytosine replaced all (MM), 5′ end half (MC), and 3′ end half (CM) cytosine bases in CC via FT-IR spectroscopy in hydrated film. Results revealed that: (i) all these triplexes have a similar hydration pattern, in which water molecules probably bound in the N₇ sites of adenines and guanines in the Crick-Hoogsteen groove, and to the methyl group of thymidines in the Watson-Hoogsteen groove. There are also some bound water molecules found at the O₂ sites of thymines in both Watson-Crick and Crick-Hoogsteen grooves, (ii) In the CC·AG6 triplex the S-type sugars are always dominant in all hydrated states, whereas in MM·AG6 triplex the relative population of the N-type sugars is very close to that of the S-type between 86% and 66% of humidity. Furthermore, the sugar conformation in two partially modified triplexes (CM·AG6, and MC·AG6) are dominant by the N-type at lower humidity. This phenomenon might reflect that the degree of bound water varies among the binding sites of bases, (iii) The effect of introducing a methyl group on cytosine is to generates spine of hydrophobic region in MM (MC and MC). The enlarging hydrophobic area not only increase the stability in solution, and also the stability in sodium hydrated films of the pyrimidine/purine/pyrimidine hairpin triplexes.     

New Low Molecular Weight Glycopeptide containing Triglucosylcysteine in Human Erythrocyte Membrane

ALTHOUGH protein composition of the erythrocyte membrane has been studied extensively¹, little if anything is known about membrane peptides. Among a variety of chemical agents used for the solubilization of the membrane, solutions of greater than 0.8 M NaCl have been shown to dissolve 50% of membrane proteins^2,3. As we were interested in the composition of the outer surface of the membrane, we have investigated whether lower concentrations of NaCl, which would not cause haemolysis of intact cells, would solubilize certain membrane components.     

Human Erythrocyte Receptor of l-Fucose-specific Lectin Produced by Streptomyces sp.

L-Fucose-specific lectin produced by Streptomyces no. 16-3 (SFL 16-3) was labeled with N- succinimidyl-[2, 3-³H]-propionate to quantitatively investigate its binding to human erythrocytes. The binding inhibition by sugars was competitive, and 5mM L-fucose or 20 mM d-mannose completely inhibited the binding. Among plant lectins, Lotus tetragonolobus, Ulex europeus I, soybean and wheat germ lectin showed competitive inhibition. The association constant and the average number of binding sites for human blood group O erythrocytes were approximately 3 × 10⁷ M^-1 and 1 × 10⁶ cell^-1, respectively. Trypsinization of erythrocytes preferentially increased the number of binding sites for human A and B erythrocytes but not for O erythrocytes.

Membrane components were extracted from human B and O erythrocytes and their binding activity for SFL 16-3 was tested using the hemagglutination-inhibition assay. Poly(glycosyl)-ceramide was the predominant receptor and its fucosyl residue was essential for binding. The crude glycoprotein fraction showed only slight inhibition activity.     

Effects of High Pressure on Glucose Transport in the Human Erythrocyte

The effects of raised hydraulic pressure on D-glucose exit from human red cells at 25 degrees C were determined using light scattering measurements in a sealed pressurized spectrofluorimeter cuvette. The reduction in the rates of glucose exit with raised pressure provides an index of the activation volume, deltaV++ (delta ln k/deltaP)(T) = -deltaV++/RT. Raised pressure decreased the rate constant of glucose exit from 0.077 +/- 0.003 s(-1) to 0.050 +/- 0.002 s(-1) (n = 5, P < 0.003). The Ki for glucose binding to the external site was 2.7 +/- 0.4 mm (0.1 MPa) and was reduced to 1.45 +/- 0.15 mm (40 MPa), (P < 0.01, Student's t test). Maltose had a biphasic effect on deltaV++. At [maltose] <250 microM, deltaV++ of glucose exit increased above that with [maltose = 0 mM], at >1 mm maltose, deltaV++ was reduced below that with [maltose = 0 mM]. Pentobarbital (2 mM) decreased the deltaV++ of net glucose exit into glucose-free solution from 30 +/- 5 ml mol(-1) (control) to 2 +/- 0.5 ml mol(-1) (P < 0.01). Raised pressure had a negligible effect on L-sorbose exit. These findings suggest that stable hydrated and liganded forms of GLUT with lower affinity towards glucose permit higher glucose mobilities across the transporter and are modelled equally well with one-alternating or a two-fixed-site kinetic models.