Search

A policy regarding rapid response to chemotherapy overdoses was developed by the authors in an attempt to minimize morbidity and mortality. The parameters of a chemotherapy overdose were defined to promote early recognition of an overdose incident. Resources needed to guide potential therapeutic interventions and required monitoring were developed. The policy defines the immediate actions to be taken in the event of a chemotherapy overdose. The availability of a chemotherapy overdose policy provides an enhanced level of safety for patients by ensuring that appropriate treatment is initiated without delay. The development of the policy was in response to the reporting of a tragic error at another institution. Healthcare providers must recognize and address potential areas of vulnerability to maximize patient safety.

Problem: Factors regulating the development, growth, and differentiation of endometrial cells of endometriotic lesions are poorly understood. To investigate the paracrine-autocrine regulation of ectopic endometrial cell growth, the expression of IGF-I and IGF-II were studied., Method: Tissue specimens of eutopic and ectopic endometria were obtained from eight patients with endometriosis at laparoscopy and from the endometria of 14 women without endometriosis as controls. They were tested for the expression of IGF-I and IGF-II by immunohistochemical analysis., Results: Immunohistochemical study for IGF-I in controls showed a more intense staining during the proliferative phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis a reduction in the staining was observed, whereas in epithelial cells of fibrotic peritoneal adhesions an intense immunostaining for IGF-I was observed. Immunohistochemical study of IGF-II in controls showed a more intense staining during secretory phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis, a reduction in the staining was observed, whereas in epithelial cells of fibrotic peritoneal adhesions an intense immunostaining for IGF-I was observed. Immunohistochemical study of IGF-II in controls showed a more intense staining during secretory phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis, a reduction in the staining was observed, whereas in epithelial cells of ovarian lesions and fibrotic peritoneal adhesions, no immunostaining for IGF-II was observed., Conclusions: In endometriosis there is an alteration of mechanisms regulating cell proliferation and differentiation.

Expression of the chicken globin genes is regulated in part by competition between the betaA-globin and epsilon-globin promoters for the enhancer found between the genes. To understand the determinants of the enhancer-promoter interaction in stage-specific regulation, the functional elements of the embryonic chicken epsilon-globin promoter were characterized. In vitro assays demonstrated that: (a) the TATA motif at -30 bound GATA-1, (b) Sp1 bound to an element centered at -54, and (c) both Sp1 and another factor, designated CACCC (which appears related to erythroid Krüppel-like factor, EKLF) bound in the -120 to -128 region. The functions of these motifs were tested using transient expression in embryonic erythroid cells. In the absence of the enhancer, promoter point mutants showed that the TATA, Sp1, and CCAAT motifs (but not the CACCC motif) contributed to promoter activity. In contrast, in the presence of the enhancer, all four motifs (including the CACCC motif) contributed to transcription. Developmental regulation of the enhancer activity was observed, with enhancement decreasing sharply from 185-fold at 4 days (cells expressing epsilon-globin) to 16-fold at 10 days (when epsilon-globin is no longer expressed). Taken together, the data suggest that multiple transcription factors contribute to promoter-enhancer interaction and the developmental regulation of epsilon-globin expression, with EKLF-like factors having an especially important role. Regulation of stage specificity occurs at the level of enhancer/epsilon-promoter interaction, even in the absence of competition, and is not simply a property of the enhancer or promoter in isolation.

Problem: The development of gestational trophoblastic tumors (GTT), in which genetic factors are strongly involved, is a rare event. To test the possibility that gene(s) linked to the Major histocompatibility Complex (MHC) may have a role in both embryo growth and tumor development, the HLA typing was performed on patients affected by GTT and on their partners., Method: The study group of sixteen couples, in which the women were affected by an invasive mole or choriocarcinoma, and the control group of thirty normal fertile couples without history of spontaneous abortion or GTT were typed for class I and class II HLA antigen., Results: The results showed no differences in single HLA-A and B antigen frequency between GTT couples and controls. In HLA-DR, locus an increased frequency of DR-6 antigen was observed (p < 0.05). No differences were observed in the frequency of number of antigens shared. When considering the single locus no differences were found in the sharing of the antigens of the A and B locus, while the frequency of antigenic sharing for DR locus was significantly higher in GTT couples with respect to controls (p < 0.025). Furthermore a higher frequency of Bw35-DR5 antigenic combination was found in GTT partners than in controls (P < 0.02)., Conclusions: These data represent a confirmation of the existence of a MHC linked gene(s) influencing the GTT development.

Problem: To determine if human leukocyte antigens (HLA) play any role in the aetiology of recurrent spontaneous abortion (RSA), a substantial group of RSA couples were studied, and their reproductive performances in a 3-year follow-up recorded., Methods: HLA typing was performed for HLA-A, -B, and DR antigens in both partners of 75 couples with unexplained RSA, and compared with a control group of 30 fertile couples that never experienced abortion. A further 57 couples of this group were studied for their reproductive performance in a 3-year follow-up, and subdivided into three subgroups: 1) couples that achieved successful pregnancy during the follow-up, and subdivided into three subgroups: 1) couples that achieved successful pregnancy during the follow-up; 2) couples that experienced abortion and no livebirth during the follow-up; and 3) couples that experienced infertility during the follow-up., Results: There were no significant differences for antigen frequency in all the different HLA loci, and HLA antigen sharing between all the RSA couples and controls. Significant increase of sharing for HLA-DR locus was observed in the couples that aborted during the follow-up with respect to the couples that achieved livebirth and controls (P < 0.03 and P < 0.02 respectively), and significantly increased frequency of B44, DR5 antigen combination in the same comparison (P < 0.03). No significant differences were observed in terms of the interval between conceptions in couples without antigen sharing with respect to couples with 1, 2 or more antigens shared, and antigen sharing in Locus A, B or DR., Conclusions: The results suggest that gene(s) disadvantageous for reproduction may exist between the HLA-B and -DR chromosomal region which influences the pregnancy outcome in RSA couples, and that HLA-antigen sharing itself does not influence the outcome.

To investigate the role of the 'male factor' in the pathogenesis of recurrent spontaneous abortion (RSA), especially sperm morphology abnormalities, 120 previously selected couples with unexplained RSA were studied for sperm parameters retrospectively and prospectively. The patients were subdivided into three subgroups, depending on their reproductive outcome during the 3 years of follow-up study: (i) 48 RSA couples who achieved a successful pregnancy; (ii) 39 RSA couples who experienced further abortions, and (iii) 33 RSA couples who experienced infertility during the follow-up period. A semen analysis was performed twice at the time of inclusion in this study, and twice again during the 3 year follow-up period. No significant differences in semen parameters were observed between RSA males and fertile controls. Instead, significant differences were observed between the group of RSA couples who experienced infertility during the follow-up and the other two groups (RSA couples who achieved successful pregnancy and RSA couples who experienced miscarriages and no live birth during the follow-up) for sperm concentration (P < 0.01 and P < 0.01 respectively), sperm motility (P < 0.01 and P < 0.01 respectively) and sperm morphology abnormalities (P < 0.01 and P < 0.01 respectively). Sperm morphology abnormalities do not seem to be involved in determining RSA; instead, they are an aetiological factor in determining infertility in patients, along with the other semen parameters, in the RSA couple's subsequent reproductive life. Semen analysis is an important test in the clinical management of RSA couples.

The DNA sequence of the Gallus gallus (chicken) beta-globin cluster was completed and analyzed. This G + C-rich region is 23.7 kb in length and includes the rho-, beta H-, beta A-, and epsilon-globin genes, the enhancer found between the beta A and epsilon genes, and three upstream DNase I hypersensitive sites. The CpG dinucleotides are nonrandomly distributed, being present at an increased relative frequency near the promoters and upstream hypersensitive sites. The cluster has an unusually low TA dinucleotide frequency. The upstream hypersensitive sites (5'HS1, 5'HS2, and 5'HS3) contain DNA sequence motifs recognized by erythroid transcription factors. However, no significant sequence similarity was found among the upstream hypersensitive sites and the beta A/epsilon enhancer. The G. gallus upstream site sequences were not similar to the upstream sites of the mammalian globin clusters, probably due to the small size of the functional regions and large evolutionary distance between the classes. The avian cluster evolved by gene duplication from an ancestor beta-globin gene, first producing the epsilon and the rho/beta H/beta A ancestor genes, then the rho and the beta H/beta A ancestor genes, and finally the beta H- and beta A-globins. Four probable gene conversions can be documented: beta A to beta H, epsilon to beta H, and rho/epsilon (twice). The cluster shows a massive overrepresentation of a non-LTR retrotransposon, CR1, which accounts for 16% of the DNA. We suggest that the locus is a preferred site for CR1 insertion.

Proteins are required for the efflux of heme from mitochondria and liposomes. The efflux from liposomes is independent of the heme-binding affinity of the protein (Biochem. 23:3715, 1984). We tested whether heme-binding proteins increase efflux of newly synthesized heme from structurally and functionally intact rat liver mitochondria. Mitochondria whose heme was labeled with 14C-delta-aminolevulinic acid, were incubated in the presence of glutathione transferases (GSTs), serum albumin (RSA) or heme-binding protein (HBP), all from the rat. HBP caused a 6-8 fold increase in efflux of newly synthesized heme as compared to that effected by RSA or GSTs. This result indicates that heme efflux from intact mitochondria, unlike that from liposomes, depends on the type of protein present and that HBP may specifically facilitate heme efflux from mitochondria.

Kinetic analysis of transferrin receptor properties in 6-8 day rat reticulocytes showed the existence of a single class of high-affinity receptors (Kd 3-10 nM), of which 20-25% were located at the cell surface and the remainder within an intracellular pool. Total transferrin receptor cycling time was 3.9 min. These studies examined the effects of various inhibitors on receptor-mediated transferrin iron delivery in order to define critical steps and events necessary to maintain the functional integrity of the pathway. Dansylcadaverine inhibited iron uptake by blocking exocytic release of transferrin and return of receptors to the cell surface, but did not affect transferrin endocytosis; this action served to deplete the surface pool of transferrin receptors, leading to shutdown of iron uptake. Calmidazolium and other putative calmodulin antagonists exerted an identical action on iron uptake and receptor recycling. The inhibitory effects of these agents on receptor recycling were overcome by the timely addition of Ca2+/ionomycin. From correlative analyses of the effects of these and other inhibitors, it was concluded that: (1) dansylcadaverine and calmodulin antagonists inhibit iron uptake by suppression of receptor recycling and exocytic transferrin release, (2) protein kinase C, transglutaminase, protein synthesis and release of transferrin-bound iron are not necessary for the functional integrity of the iron delivery pathway, (3) exocytic transferrin release and concomitant receptor recycling in rat reticulocytes is dependent upon Ca2+/calmodulin, (4) dansylcadaverine, dimethyldansylcadaverine and calmidazolium act on iron uptake by interfering with calmodulin function, and (5) the endocytotic and exocytotic arms of the iron delivery pathway are under separate regulatory control.

Marrow aspirates from sickle cell patients were examined without prior deoxygenation and revealed the presence of a variable proportion (10-30%) of sickled red cells and reticulocytes. The main feature of sickled red cells was the presence of 17.6 nm fibres arranged in hexagonal order, and to a lesser extent in square or rectangular array, to form bundles of varying size and compactness which occupied the entire cytoplasm. The sickling pattern in reticulocytes was more variable. Some reticulocytes contained highly-ordered bundles of 17.6 nm fibres whose structure and distribution was identical to that in red cells from whiich they could be distinguished only by their content of organelles. Many reticulocytes exhibited less-organized fibre patterns ranging from localized aggregates to poorly-ordered regions of short fibres and filaments lacking apparent preferential orientation. In these cells, the bulk of the cytoplasm was not polymerized. Haemoglobin polymerization in reticulocytes led to entrapment of ribosomes in concentrated foci among the fibres. Frequently, iron-laden mitochondria were associated with sickled reticulocytes. The variation in pattern of sickling seen in reticulocytes is attributed to possible differences in concentration of Hb S. Correlative studies have shown that fibres were not induced by fixation procedures. Marked phagocytic degradation of sickled cells by macrophages was observed. The results are interpreted to indicate the possible intramedullary phagocytosis of red cells and reticulocytes, predisposed to sickling in the marrow. However, the data are also consistent with the removal of sickled elements from the circulating blood.

Intracellular ferritin in newt (Triturus cristatus) erythroblasts was accessible to the chelating effects of EDTA and pyridoxal phosphate. EDTA (0.5-1 mM) promoted release of radioactive iron from ferritin of pulse-labelled erythroblasts during chase incubation, but its continuous presence was not necessary for ferritin iron mobilization. Brief exposure to EDTA was sufficient to release 60-70% of ferritin 59Fe content during ensuing chase in EDTA-free medium. EDTA also suppressed cellular iron uptake and utilization for heme synthesis, but these activities were restored upon its removal. Pyridoxal-5'-phosphate (0.5-5 mM) also stimulated loss of radioactive iron from ferritin; however, ferritin iron release by pyridoxal phosphate required its continued presence. Unlike EDTA, pyridoxal phosphate did not interfere with iron uptake or its utilization for heme synthesis. Chelator-mobilized ferritin iron accumulated initially in the hemolysate as a low-molecular-weight component and appeared to be eventually released into the medium. No radioactive ferritin was found in the medium of chelator-treated cells, indicating that secretion or loss of ferritin was not responsible for decreasing cellular ferritin 59Fe content. Moreover, there was no transfer of radioactive iron between the low-molecular-weight component released into the medium and plasma transferrin. These results indicate that chelator-released ferritin iron is not available for cellular utilization in heme synthesis and that ferritin iron released by this process is not an alternative or complementary iron source for heme synthesis. Correlation of these data with effects of succinylacetone inhibition of heme synthesis and with previous studies indicates that the main role of erythroid cell ferritin is absorption and storage of excess iron not used for heme synthesis.

1. Hemolysates of newt (Triturus cristatus) red cells contain four distinct hemoglobin species which have been observed consistently both in individual animals and pooled samples. 2. Hemoglobin heterogeneity in the species arises from existence of multiple hemoglobulins, with no indication of genetic polymorphism. 3. While the relative proportions of the different hemoglobins may vary in different samples, HbII is usually the most abundant, with HbIII and HbIV constituting most of the remainder of the total hemoglobin complement. HbI never exceeds 3-5% of the total hemoglobin. 4. Neither the electrophoretic migration nor the anion exchange properties of the four hemoglobin species are altered by conversion of oxyhemoglobin to the cyanmet derivative, excluding artifacts due to different oxidation states of iron. 5. The average molecular weight of newt total hemoglobulin is 67,182 with no indication of hemoglobin polymers. 6. The use of sulfhydryl-reducing agents (mercaptoethanol, dithiothreitol) as a precaution against aggregation results in extensive degradation of newt hemoglobin through a process similar to "coupled oxidation" by ascorbate. 7. The degradative effects of sulfhydryl-reducing agents on newt hemoglobin suggest that these reagents be used cautiously in any hemoglobin analysis.

These studies assessed the fate and localization of incoming iron in 6-8-day rat reticulocytes during inhibition of heme synthesis by succinylacetone. Succinylacetone inhibition of heme synthesis increased iron uptake by increasing the rate of receptor recycling without affecting receptor KD for transferrin, transferrin uptake, or total receptor number. Its net effect was to amplify the number of surface transferrin receptors by recruitment of receptors from an intracellular pool. Despite increased iron influx in inhibited cells, only 2-4% of total incoming iron was diverted into ferritin. The majority of incoming iron (65-80%) in succinylacetone-inhibited cells was recovered in the stroma, where ultrastructural and enzymic analyses revealed it to be accumulated mainly in mitochondria. Intramitochondrial iron (70-75%) was localized mainly in the inner membrane fraction. Removal of succinylacetone restored heme synthesis, utilizing iron accumulated within mitochondria for its support. Thus, inhibition of heme synthesis in rat reticulocytes results in accumulation of incoming iron in a functional mobile intramitochondrial precursor iron pool used directly for heme synthesis. Under normal conditions, there is no significant intracellular or intramitochondrial iron pool in reticulocytes, which are therefore dependent upon continuous delivery of transferrin-bound iron to maintain heme synthesis. Ferritin plays an insignificant role in iron metabolism of reticulocytes.

Pulse-chase analysis of newt (Triturus cristatus) erythroblasts has shown that ferritin is not a primary source of iron for heme synthesis. During chase incubation with and without non-radioactive plasma iron in the medium, no transfer of 59Fe from ferritin to hemoglobin was detected although the integrity of heme synthesis was maintained. In puromycin-inhibited cells where iron uptake was drastically curtailed, heme synthesis continued to occur, though at reduced levels; incorporation of 59Fe from the plasma appeared initially in heme and hemoglobin without any prior labelling of ferritin. These results indicate that ferritin is neither an obligatory iron intermediate in heme synthesis nor a cytosolic transport molecule involved in mobilization of iron from the transferrin-receptor complex. The most likely role for erythroid ferritin is storage of excess iron.

Erythropoietic cells of 5 species, including man, contain endoplasmic reticulum present as individual cisternae or tubules scattered throughout the cytoplasm of all stages except mature RBCs. The endoplasmic reticulum is mainly agranular but occurs frequently as a variant of granular ER which is characterized by an asymmetrical and irregular distribution of ribosomes along one cytoplasmic face. In most cells, the endoplasmic reticulum occurs in close proximity to mitochondria or the plasma membrane , suggesting that the organelle may be involved in functions related to these structures, e.g. haem biosynthesis. Endoplasmic reticulum is more abundant in early than in late erythroid cells. Its exact role in RBC development is unclear. Since endoplasmic reticulum could account for 'plasma membrane-bound ribosomes' reported in lysed reticulocytes, studies were performed which ruled out this possibility and which suggested that such ribosomes were an artifact of the lysing conditions. Hypotonic lysis in less than 20 vol. of magnesium-containing buffers yielded ghosts variably contaminated by ribosomes and other structures. Lysis of reticulocytes in 20-30 vol. of magnesium-free buffer or homogenization of whole cells or crude membrane fractions in hypotonic buffer removed virtually all contaminating ribosomes from the purified membrane fraction.

Energy-dispersive X-ray analysis has been performed on marrow sideroblasts obtained from 10 patients with sideroblastic anaemias or erythroleukaemia (six primary refractory sideroblastic anaemia, two pyridoxine-responsive, one secondary sideroblastic anaemia, two erythroleukaemia). Irrespective of the nature of the disorder associated with the presence of sideroblasts, X-ray analysis of siderotic mitochondria consistently revealed the presence of iron and phosphorus with the average Fe/P intensity ratio measuring 1.4-1.5. Other elements variably detected within siderotic mitochondria included calcium, lead, potassium and zinc. Variation in the presence of these latter elements was detected not only between different patients, but also within different samples taken at different times from a single patient and even among different cells of the same sample. Despite the detection of lead in siderotic mitochondria of a significant number of patients (five out of seven), there was no clinical evidence of lead toxicity. The elemental composition of the intramitochondrial deposits in sideroblasts was distinct from that of ferritin or haemosiderin and probably consists of ferric phosphate, possibly, ferric orthophosphate (FePO4).

The effect of changes in cellular iron metabolism on the surface expression of transferrin receptors (TfR) was examined in primary cultures of hepatocytes from adult rats. Untreated control hepatocytes exhibited a single class of high affinity receptors for transferrin (KD = 40 nM), with approximately 17,000-18,000 receptors per cell. Following 24 h of treatment with the iron chelator, desferrioxamine, or with succinylacetone, an inhibitor of heme synthesis, the number of TfR at the cell surface was increased severalfold, with no significant change in receptor affinity (KD) for transferrin. When combined, the enhancing effects of the two agents were additive. Inhibition of protein synthesis by cycloheximide abolished the increase in TfR expression mediated by either agent. Hemin decreased surface TfR expression and counteracted the enhancing effects of desferrioxamine or succinylacetone on TfR expression. These results indicate that, under the culture conditions employed, 1) iron deficiency induces an increase in surface TfR and 2) modulation of the receptor population is mainly dependent on de novo synthesis of TfR.

Newts (Triturus cristatus) made anemic with acetylphenylhydrazine (APH) fail to regenerate erythrocytes (RBC's) immediately and exhibit a latent period of 1.5-2 wk during which animals lack RBC's and are aplastic. With the establishment of erythroid regeneration at 10-14 days, relatively homogeneous populations of successive erythropoietic stages occur in the blood. This feature makes possible biochemical analyses of events in early, intermediate, and late developmental stages, respectively, each of which can be obtained in vivo with minimal contamination by other stages. Previous studies have described a primitive cell population referred to as "erythroid precursor cells" (EPC's) which precedes the appearance of definitive erythroid elements. The present studies show that EPC's and early erythroid cells are engaged mainly in ribosomal production, including synthesis of rRNA and ribosomal proteins. Moreover, EPC's and early erythroid cells also synthesize tRNA and a presumed Hb-mRNA which has been identified by its sedimentation rate at 9-12 s and its content of polyadenylic acid. In intermediate stages, there occurs a fourfold decrease in the level of RNA synthesis and, while rRNA continues to be formed, there is a disproportionate accumulation of the two major cytoplasmic rRNA species in favor of the large ribosomal subunit RNA. In late developmental stages, the level of RNA synthesis is markedly diminished with little or no evidence of formation of defined RNA classes. Correlated radioautographic and biochemical studies with radioactive delta-aminolevulinic acid and leucine indicate that EPC's and other early erythroid elements synthesize not only hemoglobin but also ferritin and ribosomal proteins. It is concluded that: (a) erythroid RNA synthesis is most pronounced in the early developmental stages, being manifested predominantly by rRNA production but including tRNA and Hb-mRNA; (b) intermediate developmental stages show both "ribosomal wastage" and decreased growth rate, marking a pivotal point between the transcriptional activities of early stages and translational activities of late stages; (c) EPC's represent a cell population already committed to RBC formation and are excluded from a role as the pluripotential stem cell.

Electron microscopic studies of bone marrow aspirates obtained from patients with homozygous sickle cell anaemia (HbSS) were fixed immediately without attempts to deoxygenate the samples. Erythroblasts and normoblasts in these preparations were devoid of haemoglobin polymers or other indications of sickling. Furthermore, the nucleated erythroid cells from sickle-cell patients presented an ultrastructural morphology indistinguishable from that of identically-processed erythroid cells in marrow samples from normal human volunteers. This report presents a description of the ultrastructural features of pronormoblasts and normoblasts in normal and sickle-cell marrows and stresses the essentially normal appearance of nucleated erythroid elements in sickle cell anaemia. Exposure of sickle-cell marrow aspirates to nitrogen at 37 degrees C for 30 min resulted in haemoglobin polymerization in most erythrocytes and reticulocytes but only in 10-20% of the nucleated erythroid cells. Haemoglobin polymers in the form of intertwining fibre meshworks were observed in reticulocytes, orthochromatic and polychromatophilic normoblasts, but were absent in basophilic normoblasts and pronormoblasts. The results suggest that the concentration of haemoglobin in intramedullary normoblasts may be the limiting factor determining the predisposition of these cells to undergo sickling as well as the pattern of haemoglobin aggregation. Under the physiological conditions prevailing in the marrow, haemoglobin concentration in normoblasts may be insufficient to result in aggregation and polymerization.

In this paper we describe the de novo appearance of azurophilic giant granules and rod-shaped inclusions in monocytes and macrophages grown from the peripheral blood and bone marrow of a patient with acute monocytic leukemia; no such inclusions were evident in direct smears of the patient's peripheral blood or bone marrow. The cytochemical profile of the inclusions, their origin in mononuclear phagocytes only, and their development exclusively in vitro suggest that they are distinct from Auer rods, Chediak-Higashi-like giant granules, and other abnormal azurophilic inclusions previously described in patients with acute non-lymphocytic leukemia.

Rabbit antisera to newt (T. cristatus) globin were produced by repeated injections of globin and antiglobin antibodies purified by chromatography on globin-Sepharose 4B. Ouchterlony and SDS PAGE analysis indicated that the material eluted from the affinity column was rabbit IgG. The antiglobin antibodies tested by immunodiffusion and ELISA cross-reacted with native hemoglobin and globin from T. cristatus and to varying extents with globins of N. viridescens, R. pipiens and X. laevis, but not with human globin. The degree of cross-reactivity appeared to parallel the evolutionary relatedness of these species, suggesting common antigenic determinants among globins of various vertebrate species.

Rat peritoneal macrophages are capable, in vitro, of processing and releasing iron derived from phagocytosed, immunosensitized red cells. From 20% to 60% of the red cell iron can be returned to the culture medium in 24 h, with resident macrophages more active than inflammatory, peptone-induced macrophages. When apotransferrin is present in the culture medium, from 39% to 72% of iron released from macrophages is bound to the protein, with most of the remainder in a ferritin-like form. No distinct preference of released iron for either site of transferrin could be observed. The absence of apotransferrin depresses iron release only slightly, with much of the iron then released in a form readily available to the protein in vitro. Pronase treatment of macrophages, which abolishes their ability to bind transferrin, depresses iron release no more than 10-15%. It appears, therefore, that binding of apotransferrin to macrophages may not be essential for iron excretion by the cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.

The fine structure of the erythrocyte during development in rabbit and human fetal liver has been studied. A morphologic description of representative erythropoietic cells and their relationship to the hepatic parenchyma is presented. Erythrocyte development was accompanied by a decrease in nuclear and cell size, fragmentation and eventual loss of nucleoli, and progressive clumping of chromatin at the nuclear margin. Mitochondria, endoplasmic reticulum, and Golgi elements decreased in size or abundance and eventually disappeared. Ribosome concentration initially increased, but subsequently diminished as the cytoplasm increased in electron opacity, probably through the accumulation of hemoglobin. Similar dense material, interpreted to be hemoglobin, infiltrated the nuclear annuli and, in some cases, appeared to extend into the interchromatin regions. There was a marked decrease in the number of annuli of the nuclear envelope. Possible relationships between nucleus and cytoplasm and of RNA to hemoglobin synthesis are discussed. In rabbits, erythroid and hepatic cells were separated by a 200 to 400 A space limited by the undulatory membranes of the respective cells. Membranes of adjacent erythropoietic cells were parallel and more closely apposed (100 to 200 A). In humans, relationship between various cells exhibited wide variation. Ferritin particles were observed within forming and formed "rhopheocytotic" vesicles.

A cytochemical study of the relationship between RNA synthesis and hemoglobin synthesis has been performed on splenectomized newts, Triturus viridescens. Employing radioautography, labeled cytidine was incorporated into the RNA of the early developmental stages but was not incorporated in the later stages. Labeled leucine was incorporated into the cellular protein of all stages except mature erythrocytes but was incorporated at a higher level in the later stages. Microphotometric measurements of azure B binding to cytoplasmic RNA revealed a sharp initial increase between the stem cell and proerythroblast followed by a rapid decrease between the basophilic and polychromatophilic stages. The loss of cytoplasmic RNA became more gradual in the late stages and, in the mature erythrocyte, little or no cytoplasmic RNA could be detected. Measurements of cytoplasmic total protein, using fast green staining at pH 2.0, and of heme showed that both curves increased similarly with development, indicating net hemoglobin synthesis. The results are compatible with the hypothesis that, as the stem cell differentiates along erythrocytic lines, a stable "messenger" RNA specifying the production of a given type or types of hemoglobin is formed. This complex probably becomes associated with ribosomal RNA and is retained throughout the process of RBC differentiation.

Studies of newt (Triturus or Diemictylus viridescens) erythropoietic cells showed that DNA synthesis and mitosis normally occur throughout most of the developmental process. Mitotic divisions were found in all immature precursor stages from the proerythroblast to the highly hemoglobinized reticulocyte. Mitoses were absent in mature erythrocytes. Radioautographic examination of thymidine-(3)H incorporation into DNA revealed that all erythroid cells except the mature erythrocyte were labeled. Microphotometric measurements of Feulgen-stained smears showed that all immature stages were undergoing DNA synthesis whereas the mature erythrocyte was inactive. The results obtained from three independent methods clearly demonstrate that (a) no loss of DNA or of chromosomes occurs during erythrocytic development and (b) highly hemoglobinized and, therefore, well-differentiated cells normally do undergo DNA synthesis and mitosis.