The metamorphic switch in hemoglobin phenotype ofXenopus laevis involves erythroid cell replacement

Summary To elucidate the cellular basis of hemoglobin transition inXenopus laevis the distribution of larval and adult hemoglobins was analyzed by indirect immunofluorescence in the circulating erythrocytes during metamorphosis. In addition, the morphological characteristics as well as the capacity for synthesis of DNA and hemoglobin in the erythrocytes were followed during the same developmental period. Our quantitative analysis on the distribution of larval and adult hemoglobins suggests that they are localized in different cells. Hemoglobin transition, therefore, most likely reflects replacement of the larval erythrocyte population by new cells which are committed to adult globin synthesis. Since hemoglobin transition is not accompanied by an increase in the abundance of immature erythroid cells with active DNA synthesis, we assume that the presumptive adult erythroid cells are released into circulation at a relatively advanced stage of maturation. The decline in the synthesis of DNA and larval hemoglobin further indicates that cessation of cell renewal in the larval erythrocyte population may represent a decisive step in hemoglobin transition.     

Impact of Zn,Cu, and Fe on the Activity of Carbonic Anhydrase of Erythrocytes in Ducks

The impact of zinc, copper, and iron on the duck erythrocyte carbonic anhydrase (CA) activity and the hemoglobin content in vitro culture were studied. The increase of zinc or iron addition at a low level induced the rise of CA activity, and the CA activity was inhibited by zinc or iron at a high addition level. The duck erythrocyte CA was strongly inhibited by cupric ion. The inhibition constant of duck erythrocyte CA to cupric ion is about 3.5 μM. Carbonic anhydrase compared to hemoglobin is more sensitive to zinc and copper in the environment. These findings suggest that some characteristics of duck erythrocyte CA are different from both CAI and CAII of mammals. The increase of Fe addition below 8 μM in the minimal essential medium brought about the rise of CA activity and resulted in the maximum of CA activity exceeding that induced by Zn. It provided a new evidence for the role of ferrous ion in CA.     

Iron release, oxidative stress and erythrocyte ageing

Iron, to be redox cycling active, has to be released from its macromolecular complexes (ferritin, transferrin, hemoproteins, etc.). Iron is released from hemoglobin or its derivatives in a nonprotein-bound, desferrioxamine-chelatable form (DCI) in a number of conditions in which the erythrocytes are subjected to oxidative stress. Such conditions can be related to toxicological events (haemolytic drugs) or to physiological situations (erythrocyte ageing, reproduced in a model of prolonged aerobic incubation), but can also result from more subtle circumstances in which a state of ischemia-reperfusion is imposed on erythrocytes (e.g., childbirth). The released iron could play a central role in oxidation of membrane proteins and senescent cell antigen (SCA) formation, one of the major pathways for erythrocyte removal. Iron chelators able to enter cells (such as ferrozine, quercetin, and fluor-benzoil-pyridoxal hydrazone) prevent both membrane protein oxidation and SCA formation. The increased release of iron observed in beta-thalassemia patients and newborns (particularly premature babies) suggests that fetal hemoglobin is more prone to release iron than adult hemoglobin. In newborns the release of iron in erythrocytes is correlated with plasma nonprotein-bound iron and may contribute to its appearance.     

Determination of genome size, macrorestriction pattern polymorphism, and nonpigmentation-specific deletion in Yersinia pestis by pulsed-field gel electrophoresis.

Of 16 restriction endonucleases known to hydrolyze rare 6- or 8-base recognition sequences that were tested, only SpeI, NotI, AscI, and SfiI generated fragments of chromosomal DNA from Yersinia pestis, the causative agent of bubonic plague, of sufficient length to permit physical analysis by pulsed-field gel electrophoresis (PFGE). Of the individual bands detected after single-dimensional PFGE of these digests, the largest sum was obtained with SpeI (3,575.6 +/- 114.6 kb). Of these 41 bands, 3 were found to contain comigrating fragments, as judged by the results of two-dimensional SpeI-ApaI PFGE; addition of these fragments and the three plasmids of the species yielded a refined estimate of 4,397.9 +/- 134.6 kb for the genome. This size was similar for eight strains of diverse geographical origin that exhibited distinct DNA macrorestriction patterns closely related to their biotypes. The high-frequency chromosomal deletion known to exist in nonpigmented mutants (unable to assimilate Fe3+ at 37 degrees C or store hemin at 26 degrees C) was shown by two-dimensional PFGE analysis with SpeI and ApaI or with SfiI and SpeI to be 92.5 and 106 kb in size, respectively. The endpoints of this deletion were precise, and its size was more than sufficient to encode the eight known peptides reported to be absent in nonpigmented mutants. This deletion had not occurred (but was able to do so) in a rare mutant capable of hemin storage but not iron transport.     

小麦蓝矮植原体染色体DNA的分离

[目的]分离小麦蓝矮(WBD)植原体染色体DNA,并建立WBD植原体染色体分离纯化体系.[方法]采用差速离心和脉冲电泳(PFGE)方法富集纯化WBD植原体染色体DNA,并通过PCR和Southern blot进行检测验证,实时荧光定量PCR方法对分离纯化效果进行定量检测.[结果]脉冲电泳凝胶中出现一条大小约为650 kb的条带,经PCR检测和Southern blot分析表明该条带为WBD植原体的染色体DNA.实时荧光定量PCR检测结果表明采用差速离心与脉冲电泳结合的方法可以将WBD植原体基因组的相对拷贝数提高436.5倍.[结论]采用差速离心与脉冲电泳法结合可以有效地从感染WBD长春花中分离到纯的WBD植原体染色体DNA,WBD植原体染色体DNA大小约为650 kb.     

炎症性贫血的病因、诊断与治疗研究进展

感染、自身免疫紊乱、慢性疾病和年龄等很多原因都会导致炎症发生并发展为贫血。炎症性贫血(anemia of inflammation AI)通常为正细胞正色素性贫血,临床表现温和。系统性的铁利用、红细胞生成、红细胞生存期特征的变化导致了炎症性贫血。最佳治疗是纠正病因,然而当病因不清或难以诊断时,治疗炎症性贫血的办法就非常有限了。铁调素(Hepcidin)是近年研究铁利用调节的中心。由于铁调素定量分析技术的发展,人们逐渐认识到其在各种疾病中的作用。最近研究集中在铁调素表达调节通路,并发现了药物治疗的靶点。由于治疗上的巨大进步,分析正常血红蛋白对疾病预后的影响,就可以明确炎性疾病发生和死亡过程中贫血是否具有可逆性。     

A robust procedure for removing background damage in assays of radiation-induced DNA fragment distributions

The non-random distribution of DNA breakage in PFGE (pulsed-field gel electrophoresis) experiments poses a problem of proper subtraction of the background DNA damage to obtain a fragment-size distribution due to radiation only. A naive bin-to-bin subtraction of the background signal will not result in the right DNA mass distribution histogram. This problem could become more pronounced for high-LET (linear energy transfer) radiation, because the fragment-size distribution manifests a higher frequency of smaller fragments. Previous systematic subtraction methods have been based on random breakage, appropriate for low-LET radiation. Moreover, an investigation is needed to determine whether the background breakage is itself random or non-random. We consider two limiting cases: (1) the background damage is present in all cells, and (2) it is present in only a small subset of cells, while other cells are not contributing to the background DNA fragmentation. We give a generalized formalism based on stochastic processes for the subtraction of the background damage in PFGE experiments for any LET and apply it to two sets of PFGE data for iron ions.     

Generation of peptides by human erythrocytes: facts and artifacts

Previously reported data on peptide composition of human erythrocyte lysate were obtained under conditions that did not exclude proteolytic degradation of hemoglobin in the process of peptide isolation. Comparative chromatographic analysis of the diluted erythrocyte lysate incubated in acidic conditions with or without proteolytic enzyme inhibitors showed that several peptides earlier identified as intraerythrocyte ones in fact result from hemoglobin degradation by erythrocyte acidic protease(s) during incubation of the lysate. A rational scheme excluding postlysis proteolysis was developed for isolation of peptide fraction. Further analysis resulted in determination of structure and content of about 50 endogenous intraerythrocyte hemoglobin fragments. A primary endopeptidase splitting of alpha- and beta-globin chains followed by consecutive exopeptidase trimming of primary fragments is suggested as a degradation mechanism. The intraerythrocyte peptides were shown to differ from peptides excreted by the erythrocytes to the extracellular medium in the primary culture. It was also found that intraerythrocyte peptides cannot play the role of precursors of hemoglobin fragments present in tissue extracts.     

Effects of excessive copper intake on hematological and hemorheological parameters

Copper plays an important role in the structure and function of metalloproteins and in the absorption of iron. The present study deals with the effects of excessive copper intake on hematological and hemorheological parameters. Drinking water containing 250 μg/mL copper for a period of 9 wk, Wistar albino rats showed increased erythrocyte count, blood viscosity, and hematocrit values (p<0.05) and lower hemoglobin (p<0.05) than controls fed a normal diet. The two groups also had differences in the erythrocyte deformability index. The results suggest that excessive copper intake results in hematological and hemorheological changes affecting both the protein content of the erythrocyte membrane and heme synthesis.     

Hemoglobin oxidation and erythrocyte hemolysis induced by a synthetic analog of alpha-tocopherol not containing an isoprenoid chain

The effect of alpha-tocopherol and its synthetic analogue which does not contain an isoprenoid chain, 2,2,5,7,8-pentamethyl-6-hydroxychroman (chromanol), on rat erythrocyte and hemoglobin has been studied. Chromanol, unlike alpha-tocopherol, induces oxidation of hemoglobin into aquomethemoglobin and causes erythrocyte hemolysis. A mechanism of the reaction has been established. It consists of two-electron reduction of haem-associated oxygen molecule. The products formed can cause oxidative membrane damage and subsequent hemolysis. The absence of similar activity of alpha-tocopherol seems to be connected with the inaccessibility of ligand sphere of hemin iron because of the presence of the isoprenoid chain. The oxidative activity of chromanol can explain the absence of E-vitamin activity in this compound.     

The red cell revisited--matters of life and death.

An erythrocyte-fractionating method combining volume and subsequent density separation is described. Iron isotope (59Fe)-validation proved this combination of methods to be complementary. By deploying HbA1c as cell age marker, obtained fractions demonstrated that circulating erythrocytes lose 20% of hemoglobin and membrane by shedding vesicles. Vesiculation from older cells proved to be facilitated by the spleen. Animal studies revealed that such vesicles are rapidly removed from the circulation by scavenger receptors on Kupffer cells with phosphatidylserine acting as the principal ligand. These studies reveal the existence of an alternative pathway of erythrocyte breakdown. This means that the premortal substrate of 20% of any erythrocyte is at our disposal. As this kind of vesiculation takes place during the entire erythrocyte lifespan, loss and sometimes reutilisation of marker substances limits the usefulness of isotope studies to the first half of the erythrocyte lifespan, thereby putting the dogmatic lifespan of 120 days into question. Furthermore, these studies add to the understanding of hemoglobin A1c (HbA1c) metabolism and the origin of the wide variation of erythrocyte parameters in peripheral blood. Removal of old erythrocytes from the circulation and from donor blood may open new ways into the treatment of both bilirubin and secondary iron overload.     

PFGE-resolved RFLP analysis and long range restriction mapping of the DNA of Arabidopsis thaliana using whole YAC clones as probes.

The cleavage patterns of 23 rare-cutting restriction endonucleases (rcREs) on high molecular weight DNA, isolated from leaves of Arabidopsis thaliana (Arabidopsis), have been analysed using pulsed field gel electrophoresis (PFGE). The DNA digested with rcREs can be used for restriction fragment length polymorphism (RFLP) analysis. We show that RFLPs are more readily identified in restriction fragments that require resolution by PFGE than in smaller restriction fragments. Taking advantage of the low dispersed repetitive DNA content of the Arabidopsis genome, whole yeast artificial chromosomes (YACs) were used as probes to PFGE resolved genomic DNA. This enabled whole YAC clones to be used as RFLP markers and long range restriction maps to be constructed. These techniques should enhance the analysis of regions of the genome of Arabidopsis (and other organisms with low levels of dispersed repetitive DNA) that are the subject of chromosome walking strategies to isolate particular loci.     

Development of large DNA methods for plants: molecular cloning of large segments of Arabidopsis and carrot DNA into yeast.

Procedures for the preparation, analysis and cloning of large DNA molecules from two different plant species are described. Arabidopsis and carrot protoplasts were used for the preparation of large DNA molecules in agarose "plugs" or in solution. Pulsed-field gel electrophoresis (PFGE) analysis of large plant DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA was at least 12 Mb. Large DNA preparations were shown to be useful for restriction enzyme analysis of the Arabidopsis genome using both frequent and infrequent cutting enzymes and for the molecular cloning of large segments of DNA into yeast using artificial chromosome (YAC) vectors. PFGE and blot hybridization analysis of Arabidopsis and carrot DNA-containing YACs indicated that both unique and highly repeated DNA sequences were represented in these libraries.     

Inhibition of the erythrocyte (Ca2+ + Mg2+)-ATPase by nonheme iron.

The erythrocyte calmodulin-stimulated (Ca2+ + Mg2+)-ATPase (CaM-ATPase), an integral membrane protein, is inhibited in different types of congenital hemolytic anemias for which oxidative processes appear as a common feature. The oxidation of hemoglobin and its degradation lead to the accumulation of ferric heme (hemin) and nonheme iron in the red cell. We have shown previously that hemin inhibits the activity of the enzyme of normal erythrocyte (Leclerc et al. (1988) Biochim. Biophys. Acta, 946, 49-56) involving an oxidation of thiol groups. The present study demonstrates that nonheme iron also inhibits the CaM-ATPase activity. In contrast with hemin, the inhibition of the enzyme induced by the nonheme treatment is prevented by butylated hydroxytoluene, a protecting agent of unsaturated phospholipid peroxidations, while dithiothreitol, a reducing agent of protein disulfide bridges, does not restore the activity of the enzyme. We conclude that nonheme iron inhibits the enzyme at least in part, through the peroxidation of phospholipids of the membrane bilayer.     

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.     

An Epidemiological Study of Salmonella enteritidis by Pulsed-Field Gel Electrophoresis (PFGE): Several PFGE Patterns Observed in Isolates from a Food Poisoning Outbreak

An epidemiological analysis of Salmonella enteritidis from a food poisoning was done using pulsed-field gel electrophoresis (PFGE) of BlnI- or XbaI-digested fragments of chromosomal DNA of isolates. S. enteritidis isolates obtained from 19 patients had identical PFGE patterns. Therefore, a strain giving the same pattern was considered to be the causative agent of this outbreak. In addition, four isolates that had different BlnI-digested PFGE patterns were obtained from three patients, suggesting that the observed variations in PFGE patterns might occur as the result of some point mutations of chromosomal DNA during growth or from the existence of several S. enteritidis strains from various sources. Subsequent PFGE analysis of continuously subcultured strains supported the former possibility. These observations indicate that PFGE analysis on multiple numbers of colonies from each patient are necessary for the epidemiologic investigation of S. enteritidis.     

Magnetic behavior of human erythrocytes at different hemoglobin states

The effect of a static magnetic field on human erythrocytes at different hemoglobin states (normal, oxidized and reduced hemoglobin) was investigated. Three different blood samples, normal, iron deficiency anemic and beta thalassemia minor, were studied. Measurements of the magnetization curves of the erythrocytes for all blood samples in all states showed diamagnetic behavior; however, oxidation was found to enhance this behavior. These measurements have also shown that the normal and iron deficiency samples in the reduced states exhibit a less diamagnetic response in comparison with the normal state. This result indicates that the reduction process gave rise to a paramagnetic component of the magnetization. Analysis of the measured paramagnetic behavior, using a Brillouin function, gave an effective magnetic moment of 8 muB per reduced hemoglobin molecule for both normal and anemic samples. This result shows that both anemic and normal blood have similar magnetic behavior and the only difference is the number of hemoglobin molecules per erythrocyte. For the beta thalassemia minor blood sample, magnetic measurements showed that both the normal and reduced states have almost the same diamagnetic behavior. However, this diamagnetic response is less than that for the normal state of the iron deficiency anemic sample. This result may indicate a low oxygen intake for the blood in the normal state for the beta thalassemia minor blood. All magnetic measurements were made using a vibrating sample magnetometer using field steps of 0.001 T from 1 T to -1 T.     

Pathogenic neisseriae can use hemoglobin, transferrin, and lactoferrin independently of the tonB locus

Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described tonB locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in Neisseria meningitidis and Neisseria gonorrhoeae. Following incubation on media containing hemoglobin, N. meningitidis IR3436 (tonB exbB exbD deletion mutant) and N. gonorrhoeae PD3401 (tonB insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb(+) variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of N. meningitidis IR3436 or N. gonorrhoeae PD3401 with chromosomal DNA from the Hb(+) variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the Neisseria Hb(+) variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously described tonB locus.