A straightforward method for analysis of highly phosphorylated inositols in blood cells by high-performance liquid chromatography.

Soluble inositol polyphosphates are found in many cells. The trisphosphate isomers, mainly inositol-1,4,5-trisphosphate, have been extensively studied because of their involvement in signal transduction. However, higher phosphorylated inositols are less frequently studied and their physiological role is poorly understood. Among these, only the myo-inositol-1,3,4,5,6-pentakisphosphate (Ins1,3,4,5,6P5), an important component of bird erythrocytes, has been intensively studied in comparative studies because it is a potent allosteric effector of hemoglobin and decreases its affinity to oxygen. We have developed a procedure for the analysis of inositol polyphosphates and other phosphate compounds in vertebrate blood cells based on a quick and accurate HPLC separation coupled to metal-dye detection. The procedure includes acid extraction of cellular phosphates, acid elimination and concentration of the extract, HPLC separation of phosphate compounds, and quantification by coupled highly sensitive metal-dye detection. The method is especially useful for analyses of highly phosphorylated inositols and for red cell comparative studies. Using the described method we have quantified Ins1,3,4,5,6P5 and the low quantities of InsP6 found in bird erythrocytes. We also identified traces of Ins3,4,5,6P4 and Ins1,3,4,6P4. Moreover, by applying the method in cultured murine macrophages, we have found changes of highly phosphorylated inositols when these cells are activated by lipopolysaccharide.     

NADH-ferrihemoglobin reductase in avian erythrocytes

The assay for NADH-ferrihemoglobin reductase (NADH-FR) was optimized for avian blood samples. In this assay the pH optimum for Japanese quail red cell NADH-FR was 5.5, which was close to the enzyme's pI. Enzyme kinetic parameters were determined for quail, chicken and turkey NADH-FR. Preparation of erythrocyte ghost-cells and subsequent fractionation showed that the enzyme was present in the plasma membrane as well as in the nuclear membrane, while Triton X-100 treatment gave a release of enzyme activity from the membrane. In the cytosolar fraction of avian red cells no NADH-FR could be detected.     

RNA polymerase in normal avian erythrocytes

Significant RNA polymerase activity was demonstrated in isolated nuclei from mature avian erythrocytes. This activity was shown to have characteristics common to mammalian systems, including sensitivity to α-amanatin. A crude fraction of RNA polymerase was solubilized from these nuclei and characterized to provide further support for the existence of the enzyme in these cells.     

Amino ketone synthesis in avian erythrocytes

1. The relative rates of synthesis of aminolaevulate and aminoacetone by particles prepared from avian erythrocytes were measured under various conditions. 2. The production of both amino ketones by fresh particles was about three times greater in anaemia caused by phenylhydrazine and acetylphenylhydrazine than in anaemia caused by removal of 20-30ml. of blood. 3. The synthesis of aminolaevulate by freeze-dried particles decreased more than that of aminoacetone in the absence of added pyridoxal phosphate, in the presence of cyanide and of tris buffer, and after preincubation of the erythrocyte particles. Other differences in the rates of synthesis of the two amino ketones were observed after (a) incubation of particles at different temperatures and (b) storage of homogenized freeze-dried particles at different pH values. 4. It is suggested that these differences in the production of the two amino ketones are due to the presence of two amino ketone synthetases or to two or more isoenzymes of aminolaevulate synthetase. 5. The metabolic significance of aminoacetone in erythrocytes is discussed.     

Nucleolar changes in maturing avian erythroblasts and erythrocytes

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.     

Free-radical mediated synthesis of enantiomerically pure, highly functionalized inositols from carbohydrates.

We report the synthesis, free-radical cyclization of precursors 1,2,7-trideoxy-7-iodo-3,4:5,6-di-O-isopropylidene-D-gluco-hept-1-enitol (1), methyl 7-O-acetyl-6-O-benzyl-8-bromo-2,3,8-trideoxy-4,5-O-isopropylidene-D-gluco-oct-2-enonate (2) and 5-O-acetyl-4-O-benzyl-6-bromo-6-deoxy-2,3-O-isopropylidene-D-glucose-O-benzyloxime (3), readily prepared from D-glucose, and some selected transformations of the carbocycles obtained from these intermediates. In compound 1 we have installed a terminal double bond and an iodide as radical acceptor and leaving group, respectively. Compounds 2 and 3 are epsilon-bromo aldehydes substituted with alpha,beta-unsaturated ester and oxime ether functions as radical traps, respectively. The tributyltin hydride mediated ring closure of these radical precursors have afforded a series of interesting, diverse and highly functionalized carbocycles which can be considered useful building blocks for the synthesis of branched-chain cyclitols, aminocyclitols and aminoconduritols. In these processes, a good chemical yield and high stereoselectivity has been found in the newly formed stereocenters. Particularly interesting has been the finding that the stereochemical outcome of the free-radical cyclization is independent of the ratio of isomers (E or Z) in oxime ether 3. These results show the power and the state of art of this strategy for the stereocontrolled synthesis of enantiomerically pure inositols from carbohydrates.     

Sugar transport in reversibly hemolyzed avian erythrocytes

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37°C, 88% of the ghosts regained their permeability barrier to l-glucose. In these ghosts, the carrier-mediated rate of entry of $\text{3-O-}\text{methylglucose}$ was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

高致病性禽流感防控难点的分析

禽流感(avian influenza,AI)是由禽流感病毒引起的一种严重危害畜牧业的急性传染病,特别是高致病性禽流感引起禽类的呼吸系统感染以及全身性败血症,死亡率极高。多年来,许多国家和地区都爆发过此病,造成巨大经济损失,而2004年亚洲爆发的H5N1亚型禽流感造成经济损失的同时还出现了众多的禽流感病毒直接感染人类、造成人员死亡病例,再一次把人类的目光转移向此病。AI抗原类型众多,变异频繁,不同的类型抗原之间无交叉反应,同时,病毒具有复杂的感染和复制机制以及复杂的传播网络等多种因素单独和,协同作用,导致高致病性禽流感防控困难。     

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose entry and exit, $\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>si</mtext>}}$, were 8 and 7 mM, respectively. The equilibrium exchange constant, $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$, and the counterflow constant, $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$, were 9 and 11 mM, respectively. The activity constant for $\text{3-O-}\text{methylglucose}$ transport, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, defined as $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, $\text{K}{}_{\mathrm{<mtext>go</mtext>}}$, was 2 mM and less than the counterflow constant, $\text{R}{}_{\mathrm{<mtext>g</mtext>}}$ (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, $\text{F}{}_{\mathrm{<mtext>g</mtext>}}$, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ($\text{K}{}_{\mathrm{<mtext>si</mtext>}}$ and $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$) for $\text{3-O-}\text{methylglucose}$ transport were significantly larger than those obtained in anaerobic cells, but the remaining two ($\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$) remained unchanged. The values, for $\text{K}{}_{\mathrm{<mtext>so</mtext>}}\text{, K}{}_{\mathrm{<mtext>si</mtext>}}\text{, B}{}_{\mathrm{<mtext>s</mtext><mtext>\; and</mtext>}}\text{R}{}_{\mathrm{<mtext>s</mtext>}}$ were 8, 26, 20 and 8 mM, respectively. The activity constant, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.     

DNA single-strand breaks in adult and embryonic avian erythrocytes

We have investigated the possibility that the reactivation rate of adult avian erythrocytes, which is slower than that of embryonic erythrocytes, after fusion with metabolically active cells, is due to a greater number of single-strand breaks (ssb) in the DNA of the former. We have assayed ssb by measuring the template activity of the erythrocyte nuclei for added Escherichia coli DNA polymerase. We have found that differences in the numbers of ssb within polymerase-accessible regions between adult and embryonic cells are within experimental error. We conclude that, unless very localized clusters of damage exist within the DNA (which would not be detectable by this or other techniques), the difference in reactivation rate is not attributable to differences in ssb numbers.     

RNA polymerases during differentiation of avian erythrocytes

DNA-dependent RNA polymerase was analysed during the terminal differentiation stages of avian erythrocytes. It was found that the mature duck erythrocyte, although quiescent in RNA synthesis, contains clearly measurable quantities of RNA polymerase B (or II). Immature polychromatic erythrocytes, derived from anemic ducks and actively synthesizing hemoglobin mRNA, additionally contain significant amounts of RNA polymerase A (or I) and C (or III) previously not detected in these cells. These latter classes of enzymes, although present, are apparently not engaged in RNA synthesis in polychromatic erythrocytes.     

Agonist-stimulated inositol phosphate metabolism in avian erythrocytes.

1. A screen for agonists capable of stimulating the formation of inositol phosphates in erythrocytes from 5-day-old chickens revealed the presence of a population of phosphoinositidase C-linked purinergic receptors. 2. If chicken erythrocytes prelabelled with [3H]Ins were exposed to a maximal effective dose of adenosine 5'-[beta-thio]diphosphate for 30 s, the agonist-stimulated increment in total [3H]inositol phosphates was confined to [3H]Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP2. After 40 min stimulation, the radiolabelling of nearly all of the [3H]inositol phosphates that have been detected in these extracts [Stephens, Hawkins & Downes (1989) Biochem. J. 262, 727-737] had risen. However, some of these increases [especially those in Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5] were accountable for almost entirely by increases in specific radioactivity rather than in mass. 3. The effect of purinergic stimulation on the rate of incorporation of [32P]Pi in the medium into the gamma-phosphate group of ATP and InsP4 and InsP5 was also measured. After 40 min stimulation, the incorporation of 32P into Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5 was significantly elevated, whereas the mass of the last two and the specific radioactivity of the gamma-phosphate of ATP were unchanged compared with control erythrocyte suspensions. 4. In control suspensions of avian erythrocytes, the specific radioactivity of the individual phosphate moieties of Ins(1,3,4,6)P4 increased through the series 1, 6, 4 and 3 [Stephens & Downes (1990) Biochem. J. 265, 435-452]. This pattern of 32P incorporation is not the anticipated outcome of 6-hydroxy phosphorylation of Ins(1,3,4)P3 [the assumed route of synthesis of Ins(1,3,4,6)P4]. Although adenosine [beta-thio]diphosphate significantly stimulated the accumulation of [3H]Ins(1,3,4)P3, and despite the fact that avian erythrocyte lysates were shown to possess a chromatographically distinct, soluble, ATP-dependent, Ins(1,3,4)P3 6-hydroxykinase activity, purinergic stimulation of intact cells did not significantly alter the pattern of incorporation of [32P]Pi into the individual phosphate moieties of Ins(1,3,4,6)P4. These results suggest that the route of synthesis of this inositol phosphate species is not changed during the presence of an agonist.     

Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.