Red Cell Nuclei Research Articles

Artefactual Suboptimal Fixation Effect to Nuclear Staining on Erythrocytes of Lutjanus kasmira (Forsskål 1775)

This research presents the unusual staining characteristic of the teleosts red blood cells–the common bluestripe snapper Lutjanus kasmira, caught at Toc Tan (Allison) Reef, Truong Sa Archipelago, Vietnam, due to side effects of the delayed staining procedure. In addition to the quantitative data measured, the results of microscopic image research showed that, on slides performed with the delayed Romanovsky staining procedure using Giemsa dye, the cell "nucleus" was not dye-stained as seen in the erythrocytes of much other fish. The AFM and SEM images of blood slides show that the structure of the nuclear zone and the nuclear membrane was destroyed. Although methanol is used as one of the common fixatives in biology, the delayed staining still causes artifactual side effects, disrupts the structure of the red blood cell nucleus and can cause confusion when studying cytogenetics.

Cardiovascular Manifestations of Systemic Lupus Erythematosus: An Overview

Systemic Lupus Erythematosus(SLE) is a multisystem autoimmune disease with variable presentations. A proposed mechanism for the etiology of SLE involves the development of autoantibodies that result from a defect in apoptosis. The specific defect involves the “find-me” (adenosine triphosphate [ATP]/uridine triphosphate [UTP]) or “eat-me” (phosphatidylserine) signals activated upon release of red cell nuclei. In the absence of apoptosis, the nuclei break down, causing inflammation and contributing to the development of autoimmunity. Many signs and symptoms of SLE are caused by either circulating immune complexes or direct effects of antibodies on cells. A genetic predisposition for SLE exists, and the concordance rate in monozygotic twins is between 25% and 70%. If a mother has SLE, her daughter’s risk of developing the disease is 1:40, and her son’s risk is 1:250. The course of SLE consists of intermittent remissions punctuated by disease flares, and organ damage often progresses over time. Pericarditis is the most frequent cardiac manifestation; it can be documented by ECG, auscultation of a friction rub, or evidence of pericardial effusion. It usually responds to anti-inflammatory therapy and infrequently leads to tamponade. More serious cardiac manifestations are myocarditis and fibrinous endocarditis of Libman-Sacks. The endocardial involvement can lead to valvular insufficiencies, most commonly of the mitral or aortic valves, or embolic events. It has not been proven that glucocorticoid or other immunosuppressive therapies improve lupus myocarditis or endocarditis. Still, it is usual practice to administer a trial of high-dose steroids and appropriate supportive treatment for heart failure, arrhythmia, or embolic events. As discussed above, patients with SLE are at increased risk for myocardial infarction, usually due to accelerated atherosclerosis, which probably results from immune attack, chronic inflammation, and chronic oxidative damage to arteries.

Simulating photoacoustic waves produced by individual biological particles with spheroidal wave functions.

Under the usual approximation of treating a biological particle as a spheroidal droplet, we consider the analysis of its size and shape with the high frequency photoacoustics and develop a numerical method which can simulate its characteristic photoacoustic waves. This numerical method is based on the calculation of spheroidal wave functions, and when comparing to the finite element model (FEM) calculation, can reveal more physical information and can provide results independently at each spatial points. As the demonstration, red blood cells (RBCs) and MCF7 cell nuclei are studied, and their photoacoustic responses including field distribution, spectral amplitude, and pulse forming are calculated. We expect that integrating this numerical method with the high frequency photoacoustic measurement will form a new modality being extra to the light scattering method, for fast assessing the morphology of a biological particle.

Ploidy effects on hatchery survival, deformities, and performance in Atlantic salmon ( Salmo salar)

The production of sterile triploid Atlantic salmon ( Salmo salar) may help address the increasing pressure on the industry to reduce potential breeding between farmed escapees and wild fish populations. However, many previous studies have observed poor performance at sea in triploid stocks (growth, survival, and deformity). This may result from poor early hatchery performance and a strong parental effect. Therefore, in the present study, two year classes (2007 and 2008) of mixed sex fish were created (10 males:10 females) to examine ploidy interactions on hatchery performance. Egg batches were divided in two at fertilisation with one group subjected to a hydrostatic pressure shock to induce triploidy. Triploid rate was confirmed at 100% in all groups, verified by red blood cell nucleus length measurements. Survival to hatch did not differ between ploidy. However, reduced survival was found to strongly correlate with gamete quality. During the hatchery phase ploidy significantly affected size at hatch, with diploids generally larger than triploids. Growth advantage of diploids over triploids was only maintained for 6 weeks post-first feeding, with triploids generally out-growing their diploid siblings by the end of the hatchery phase. Deformity prevalence in first feeding stages was generally low (mean < 2%), with no overall effect of ploidy. Our findings show that triploid salmon can perform as well if not better than their diploid siblings. The low incidence of deformity during the hatchery and freshwater phases is also a significant improvement over previous reports in triploid salmon stocks.

The First Trimester Human Placenta Is An Embryonic Hematopoietic Organ with An Unexpected Function in Primitive Erythroid Maturation.

Abstract 1508Poster Board I-531Developmental hematopoiesis satisfies the immediate needs of the embryo and provides the hematopoietic stem cells necessary for lifelong blood homeostasis. Recently, the mid-gestation mouse placenta was identified as an important definitive hematopoietic organ; data from us and others indicates that the human placenta functions analogously, harboring and potentially generating hematopoietic stem and progenitor cells. The function of the human placenta in embryonic hematopoiesis has not been addressed. We assessed the hematopoietic potential of placental tissues before the onset of feto-placental circulation and found robust de novo generation of clonogenic progenitors. Interestingly, pre-circulation progenitors were greatly enriched (69 ± 14% of total) in macrophage progenitors. Immunostaining demonstrated that these progenitors are generated in the chorionic plate. As development progresses, placental macrophages migrate to the villous stroma. Surprisingly, in the villi, placental macrophages associate closely with an unexpected population of extravascular, z-globin+ primitive erythroid cells, prompting the hypothesis that embryonic macrophages promote the maturation of primitive erythroblasts in the placenta. Concordantly, we found that human primitive erythroblasts enucleate, as has been recently demonstrated in mice. Interestingly, the first enucleated erythroid cells were found in the villous stroma; between 5-7 weeks developmental age, their relative frequency in the stroma was 2-4 fold higher than in lumens. We also observed free nuclei in the placental stroma; a similar population of ejected red cell nuclei, termed pyrenocytes, has recently been described in mouse embryos. Immunohistochemistry and FACS confirmed that these free nuclei in the placenta were pyrenocytes. Electron microscopy revealed placental macrophages containing ingested red cell nuclei. Together, this data suggests that placenta-derived macrophages promote the terminal maturation of primitive erythroblasts in the villous stroma, nominating the first trimester human placenta as a site of primitive hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Genome size, cell size, and the evolution of enucleated erythrocytes in attenuate salamanders

Within the salamander family Plethodontidae, five different clades have evolved high levels of enucleated red blood cells, which are extremely unusual among non-mammalian vertebrates. In each of these five clades, the salamanders have large genomes and miniaturized or attenuated body forms. Such a correlation suggests that the loss of nuclei in red blood cells may be related, in part, to the interaction between large genome size and small body size, which has been shown to have profound morphological consequences for the nervous and visual systems in plethodontids. Previous work has demonstrated that variation in both the level of enucleated cells and the size of the nuclear genome exists among species of the monophyletic plethodontid genus Batrachoseps. Here, we report extensive intraspecific variation in levels of enucleated red blood cells in 15 species and provide measurements of red blood cell size, nucleus size, and genome size for 13 species of Batrachoseps. We present a new phylogenetic hypothesis for the genus based on 6150 bp of mitochondrial DNA sequence data from nine exemplar taxa and use it to examine the relationship between genome size and enucleated red blood cell morphology in a phylogenetic framework. Our analyses demonstrate positive direct correlations between genome size, nucleus size, and both nucleated and enucleated cell sizes within Batrachoseps, although only the relationship between genome size and nucleus size is significant when phylogenetically independent contrasts are used. In light of our results and broader studies of comparative hematology, we propose that high levels of enucleated, variably sized red blood cells in Batrachoseps may have evolved in response to rheological problems associated with the circulation of large red blood cells containing large, bulky nuclei in an attenuate organism.

Erythrocyte senescence and haematological changes induced by starvation in the neotropical fish traíra, Hoplias malabaricus (Characiformes, Erythrinidae)

Adult specimens of traíra ( Hoplias malabaricus Bloch) were subjected to long-term starvation (30 to 240 days) and re-fed for 30 days after 90 and 240 days of food deprivation. Counting of immature erythrocytes in peripheral blood showed that erythropoiesis decreased significantly during the first 30 days of food deprivation. The results suggest that a process of senescence takes place in the pre-existent red blood cells and that the cells are not replaced during starvation. After 240 days of starvation, H. malabaricus had a significantly reduced number of red blood cells, causing changes in hematocrit and blood indices (mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration). Furthermore, during this period, the fish presented leukopenia (lymphocytopenia) and thrombocytopenia. After re-feeding, the number of leukocytes and thrombocytes recovered, but the red blood cell number remained reduced and there was a significant increase in abnormal red cell nuclei.

Genotoxic effects of cadmium chloride and azadirachtin treated singly and in combination in fish

The genotoxic effects of cadmium chloride (CdCl 2) and azadirachtin (Aza) were assessed singly and conjointly in a fish, Oreochromis mossambicus, with endpoints such as chromosome aberrations, abnormal red cell nuclei, abnormal sperm morphology, and protein content (both qualitative and quantitative) of selected tissues, namely, muscle, heart, eye, brain, gill, liver, spleen, and kidney. The primary objectives were, first, to examine if CdCl 2, a common pollutant, and Aza, a natural product of the neem plant used extensively as an ‘ecofriendly’ agent for many purposes, had any genotoxic effect of their own on nontarget aquatic organisms of economic importance; and second, if Aza could have any ameliorating effect on CdCl 2-induced genotoxicity in O. mossambicus tissues. As compared with distilled water-treated controls, both CdCl 2 and Aza induced genotoxicity in O. mossambicus, the former in greater quantity than that produced by Aza. However, Cd-induced toxicity in O. mossambicus appeared to be ameliorated to some extent by Aza.

Mosaic individuals found in genetically manipulated northern pike (Esox lucius) using flow cytometry

The purpose of this study was to investigate abnormal larvae produced in the experimental induction of gynogenetic northern pike (Esox lucius L.), using a flow cytometry method. We reported for the first time the mosaic individuals found in genetically manipulated northern pike. Two types of abnormal larvae were obtained after eggs were fertilized with UV-irradiated sperm and then exposed to heat shock treatment. One type had malformations frequently associated with haploid syndrome and were proven to be haploids. The other type had a similar body size and appearance as diploids but with curved body and a swirling swimming pattern. Individuals of the second type were proven to be mosaics containing both 1n (59.5 ± 14.7% mean ± SD) and 2n (29.7 ± 13.3%) cell populations. The ratio of 1n:2n cell populations ranged from 80.6:10.6 to 38.5:44.7. There was no significant difference in the relative nuclear DNA content and cell size between 1n and 2n cell populations of the mosaic individuals as compared to haploid cells and diploid cells, respectively. The nuclear DNA content of the northern pike was estimated to be 2.07 ± 0.06 pg (n = 10) given that rainbow trout has a red blood cell nucleus DNA content of 4.66 pg.

Description and Epizootiology of Babesia poelea n. sp. in Brown Boobies (Sula leucogaster (Boddaert)) on Sand Island, Johnston Atoll, Central Pacific

We describe a new species of piroplasm from brown boobies (Sula leucogaster) on Sand Island, Johnston Atoll National Wildlife Refuge, central Pacific. Mean parasitemia in adults and chicks was less than 1%, with the parasitemia in chicks significantly greater than in adults. There was no significant relation between the age of chicks and the degree of parasitemia. Parasitized red cells and red cell nuclei were significantly smaller than those of unparasitized cells, and infected birds appeared clinically normal. Prevalence of the parasite in chicks (54%) was significantly greater than in adults (13%), and the geographic distribution of parasitized chicks was skewed toward the eastern end of Sand Island. On the basis of morphologic characteristics, we named it Babesia poelea. The specific name is a concatenation of the Hawaiian names for dark (po'ele) and booby ('a). This is the second documentation of an endemic avian hemoparasite in seabirds from the central Pacific.

The paradox of ferrata and the fate of the extruded red cell nucleus: Two problems concerning erythropoiesis in the human

A critical analysis of the present erythropoietic pathway reveals two problems which require resolving. One is the paradox of the basophilic erythroblast, and the other is the fate of the extruded red cell nucleus. The problems can be overcome if erythropoiesis is looked at differently, and the orthochromatic normoblast considered to arise directly from a denuded stem cell nucleus as has been previously suggested. The orthochromatic normoblast extrudes its nucleus leaving behind a reticulocyte. The extruded but functionally impaired nucleus of the orthochromatic normoblast then gives rise to a polychromatic normoblast, a defective cell. The poorly made cytoplasm of the polychromatic normoblast is shed and its nucleus, now non-functional, undergoes complete dissolution into an aggregate of ultrafine particles. The theory has the advantage that the fate of the extruded red cell nucleus can be explained without having to introduce phagocytosis by macrophages and all the immunological difficulties which this entails. The new pathway does away with the basophilic erythroblast as a haemoglobin-producing cell, and it is argued that the cell instead is a ferritin storage cell, and that erythropoiesis is the result of two separate but interdependent pathways, a ferritin storage pathway and a haemoglobin production pathway. Evidence is put forward to support the new pathways.

Transcriptional properties of chick embryonic erythroid nuclei in vitro.

Chick embryo red blood cells express the embryonic globin and the rRNA genes before 5 days of development but only the adult globin genes and no rRNA after 12 days of development. We have isolated nuclei from the red blood cells of these developmental stages and allowed them to transcribe in vitro. We have analyzed the overall transcriptional properties of these nuclei, the overall activities of RNA polymerases I and II and the sequence-specific activity of RNA polymerase II in the beta-globin domain using cloned genomic hybridization probes. Among our findings are the following. 1) Erythroid nuclei of 5-day embryos are more transcriptionally active than those at 12 days. 2) RNA polymerase I is a very active at 5 days but is off by 12 days. 3) A template-independent activity which yields labeled RNA is present in the red cell nuclei of 12-day but not 5-day embryos. 4) Between 5 and 12 days of development transcriptional modulation delineates embryonic and adult beta-globin domains. 5) These domains exceed the boundaries of the genes themselves by several kilobases. 6) All transcripts which hybridize to sequences in the beta-globin gene region, including repeat sequence transcripts, are transcribed by RNA polymerase II.

An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin.

The single-stranded activity of S1-nuclease cleaves globin chromatin in red cell nuclei in specific regions. The cleavages are observed only in tissues in which the globin genes are active, and they "switch" to reflect the switching pattern of globin-gene expression in embryonic and adult red cells. The positions of the S1 cleavages in the beta- and alpha-globin chromatin correspond to the general region of known DNAase I-hypersensitive sites, but can be distinguished in detail. When DNA segments containing these regions are subcloned into pBR322 and the supercoiled molecules are treated with S1, similar sites are cleaved in the purified supercoiled (but not linear) recombinant plasmid DNA. However, the dominant S1 cutting sites are shifted in the plasmid vis-a-vis the chromatin. We believe that some aspect of DNA sequence is translated into an altered DNA structure in chromatin and that it is this altered structure that is recognized by s1 nuclease and possibly by certain chromosomal proteins. Several physical properties reflected in the S1 digestion of supercoiled plasmids suggest a mechanism for generating differences in daughter cells during development.

Cellular and subcellular localization of polyamines cytochemical methods providing new clues to polyamine function in normal and neoplastic cells.

Studies on the cellular and subcellular localization of polyamines have previously been hampered by the rapid redistribution of these organic cations during cell fractionation. Cytochemical methods selectively localizing polyamines would, hence, be important for our understanding of where polyamines are stored and where they work. With the aid of chemical characterizations of tissue extracts, biosynthesis experiments and cytochemical model experiments we show that the cytochemical formaldehyde-fluorescamine (FF) technique selectively detects the polyamines spermine and spermidine. Results obtained with the FF method concur with previous chemical observations by showing that growing and neoplastic cell populations are rich in cytochemically detectable polyamines. The FF method also localizes polyamines to certain normal secretory cell systems, including pancreatic exocrine and endocrine (insulin) cells, cells and secretion products in the ventral prostate, pituitary growth hormone cells and neurosecretory nerve endings and leucocytes. In most cell systems studied, polyamines appear to be most concentrated in cytoplasmic structures. However, in HeLa cell mitotic chromosomes and in frog red cell nuclei most polyamines seem to be chromosomes and in frog red cell nuclei most polyamines seem to be associated with chromatin. These observations raise important questions concerning both extra- and intracellular functions of polyamines.

Transcriptional regulation of hemoglobin switching in chicken embryos.

We used recombinant chicken deoxyribonucleic acid clones containing embryonic and adult beta-globin genes and "runoff" endogenous nuclear transcription to investigate the expression of embryonic and adult beta-globin genes during hematopoiesis in the developing chicken embryo. Purified, cloned deoxyribonucleic acids were digested with various restriction enzymes, separated on agarose gels, blotted to nitrocellulose, and annealed with purified nuclear [32P]ribonucleic acid synthesized in vitro from embryonic or adult red cell nuclei. Transcription of the respective globin genes was assayed by hybridization of nuclear [32P]ribonucleic acid to specific restriction fragments containing adult or embryonic coding sequences. Our results indicate that little, if any, transcription from the adult or embryonic beta-globin genes is detectable in the heterologous red cell nuclei, even under conditions in which ribonucleic acid processing probably does not occur.

Transcriptional regulation of hemoglobin switching in chicken embryos.

We used recombinant chicken deoxyribonucleic acid clones containing embryonic and adult beta-globin genes and "runoff" endogenous nuclear transcription to investigate the expression of embryonic and adult beta-globin genes during hematopoiesis in the developing chicken embryo. Purified, cloned deoxyribonucleic acids were digested with various restriction enzymes, separated on agarose gels, blotted to nitrocellulose, and annealed with purified nuclear [32P]ribonucleic acid synthesized in vitro from embryonic or adult red cell nuclei. Transcription of the respective globin genes was assayed by hybridization of nuclear [32P]ribonucleic acid to specific restriction fragments containing adult or embryonic coding sequences. Our results indicate that little, if any, transcription from the adult or embryonic beta-globin genes is detectable in the heterologous red cell nuclei, even under conditions in which ribonucleic acid processing probably does not occur.

Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNAase I

Using recombinant chicken DNA clones as probes, we have investigated the DNAase I sensitivity of chromosomal DNA regions bordering the α- and β-globin structural genes. By both a blot hybridization assay and solution hybridization, we find that regions around these globin genes are preferentially sensitive (relative to the ovalbumin gene) to DNAase I after mild digestion of isolated red cell nuclei. These regions are resistant in cells that do not express globin. The preferential DNAase I sensitivity extends to at least 8 kb on the 3′ side of the β-globin gene cluster and to 6 or 7 kb on the 5′ side, where relatively resistant DNA fragments have been identified. Using low levels of DNAase I to titrate the sensitivities of coding and adjacent noncoding regions, it was observed that coding regions are organized into a very sensitive structure, while adjacent noncoding regions are organized into a moderately sensitive structure. The blot hybridization assay has also revealed that DNAase I introduces specific, double-stranded cuts into both the α- and β-globin gene clusters. Many of these cuts are tissue-specific. Several α gene-specific sites occur toward the 3′ side of the α-coding sequences. The β sites are different in embryonic and adult red cells. In embryonic cells, the cut occurs near the 5′ end of an embryonic β gene, while in adult cells there are two cuts, one at approximately 2 kb and the other at approximately 6 kb from the 5′ side of an adult gene. Based on the observation that the general region around the origin for replication and promotors for transcription in the SV40 minichromosome is also very sensitive to specific, double-stranded scissions by DNAase I, we speculate that the specific cuts in the globin domain may be structural modifications of the chromatin that are associated with origins for DNA replication or promotors for transcription.

Hb switching in chickens

We have taken advantage of the preferential digestion of active genes by DNAase I to investigate the chromosomal structure of embryonic and adult β-globin genes during erythropoiesis in chick embryos, and in particular to examine the question of hemoglobin switching during development. DNA in isolated red cell nuclei was mildly digested with DNAase I to about 10–15 kb, purified and restricted with a variety of restriction enzymes. The DNA was then separated on agarose gels, transferred to nitrocellulose filters and hybridized with an adult-specific β-globin cDNA clone or a genomic clone containing the genes coding for both an embryonic and an adult β-globin chain. Preferential sensitivity of the respective globin genes was monitored by the disappearance of specific restriction bands after DNAase I digestion of nuclei. In embryonic red cells, both adult and embryonic β-globin genes are very sensitive to DNAase I; however, in adult erythroid lines, the embryonic β-globin gene becomes relatively more resistant but the adult gene remains highly sensitive. Controls showed that all globin genes were resistant to DNAase I in brain nuclei and nuclei from lymphoid cells. Thus the switch from embryonic to adult globin expression is associated with an apparent change in the chromosome structure of the embryonic globin gene as reflected in the gene becoming less accessible to DNAase I in adult red cell nuclei. Our results also show that the chromosomal structure of both adult and embryonic genes is altered in embryonic red cell nuclei; thus the nonexpressed globin gene (that is, the adult gene in embryonic red cells) has already been “recognized” to some degree and marked by the erythroid compartment. The sensitivity of the adult globin gene in embryonic cells may represent a “pre-activation” state of the chromosome.

The chemical nature of de Tomasi Schiff reagent. Changes of certain physical-chemical and histochemical properties during ageing.

Samples of de Tomasi's Schiff reagent were allowed to age. During this process physical, chemical and staining properties of the reagent were monitored. Certain physical and chemical properties of Schiff's reagent correlated with staining intensities. Thus a fall in the iodine titre and a rise in the pH (indicating loss of sulphur dioxide) accompanied falls of intensity of Feulgen-stained frog red-cell nuclei and of P.A.S.-stained cellulose sheet. Ageing sulphite solutions exhibited analogous physical and chemical changes. Thin layer and ion-exchange chromatography of Schiff's reagent demonstrated at lest four colourless (but colour generating) derivatives, all of which were cationic. NMR spectroscopy showed that the carbon skeletons of these compounds were simple and symmetrical. The ageing of Schiff's reagent appears to be primarily due to changes in its inorganic components. The aldehyde-reactive compounds probably differ only in the nature of the oxysulphur substituent on their central carbon atom.

Detection of chick rRNA in the cytoplasm of heterokaryons containing reactivated chick red cell nuclei

In order to demonstrate the presence of chick ribosomal RNA (rRNA) in the cytoplasm of heterokaryons containing reactivated chick red cell nuclei, it was necessary to increase the sensitivity of detection. The method devised took advantage of the fact that avian cells in general are capable of growing satisfactorily at higher temperatures than human cells. rRNA synthesis is temperature sensitive in human cells. The results show that reactivated chick red cell nuclei produce a significant amount of 28S rRNA, but much less than expected from autoradiographic studies. It is also clear that the temperature-sensitive function in the human cell cannot be complemented, even by a fully functional cell like a chick fibroblast.