Long-acting PGE2 and Lisinopril Mitigate H-ARS.

Thrombocytopenia is a major complication in hematopoietic-acute radiation syndrome (H-ARS) that increases the risk of mortality from uncontrolled hemorrhage. There is a great demand for new therapies to improve survival and mitigate bleeding in H-ARS. Thrombopoiesis requires interactions between megakaryocytes (MKs) and endothelial cells. 16, 16-dimethyl prostaglandin E2 (dmPGE2), a longer-acting analogue of PGE2, promotes hematopoietic recovery after total-body irradiation (TBI), and various angiotensin-converting enzyme (ACE) inhibitors mitigate endothelial injury after radiation exposure. Here, we tested a combination therapy of dmPGE2 and lisinopril to mitigate thrombocytopenia in murine models of H-ARS following TBI. After 7.75 Gy TBI, dmPGE2 and lisinopril each increased survival relative to vehicle controls. Importantly, combined dmPGE2 and lisinopril therapy enhanced survival greater than either individual agent. Studies performed after 4 Gy TBI revealed reduced numbers of marrow MKs and circulating platelets. In addition, sublethal TBI induced abnormalities both in MK maturation and in in vitro and in vivo platelet function. dmPGE2, alone and in combination with lisinopril, improved recovery of marrow MKs and peripheral platelets. Finally, sublethal TBI transiently reduced the number of marrow Lin-CD45-CD31+Sca-1- sinusoidal endothelial cells, while combined dmPGE2 and lisinopril treatment, but not single-agent treatment, accelerated their recovery. Taken together, these data support the concept that combined dmPGE2 and lisinopril therapy improves thrombocytopenia and survival by promoting recovery of the MK lineage, as well as the MK niche, in the setting of H-ARS.

Delineating stages of erythropoiesis using imaging flow cytometry.

Adult humans need to make 2.5million red blood cells (RBCs) every second to maintain a steady state level of 25trillion circulating RBCs. Understanding normal erythropoiesis as well as diseases that afflict the erythron, such as genetic anemias, hyperproliferative disorders, and myelodysplastic syndromes, requires a robust method to delineate erythropoietic intermediates. In order to apply the power of flow cytometry to these studies, challenges of limited immunophenotypic markers, incorporation of significant changes in morphology, and maturational changes that occur along a continuum need to be met. Imaging flow cytometry (IFC) provides a solution to address these challenges. Integration of changes in immunophenotype, loss of RNA (ribosomes), and enucleation, with morphological characteristics of cell and nuclear size, can be used to delineate erythroblasts that correlate with classical histological classifications. A protocol is described that demonstrates the basic approaches of staining panel selection, mask generation and selection of features to best sequentially refine erythroid intermediates and remove contaminating cells with overlapping immunophenotype. Ultimately erythroid cells in the murine bone marrow are divided into seven sub-populations using IFC including four erythroblasts (pro-, basophilic, polychromatophilic and orthochromatic), the pyrenocyte, which contains the eliminated nucleus, the enucleated reticulocyte and the mature RBC.

Serglycin proteoglycan synthesis in the murine uterine decidua and early embryo.

This study has explored the localization and synthesis of the serglycin proteoglycan in the murine embryo and uterine decidua during midgestation. Embryos in deciduae were subjected to in situ hybridization with cRNA probes and to immunohistochemical detection with a specific antibody against murine serglycin. Adherent decidual cell cultures were prepared from freshly isolated deciduae. Proteoglycan biosynthesis was investigated by labeling intact deciduae and decidual cultures with (35)S-sulfate. Serglycin mRNA was detected by in situ hybridization throughout the mesometrial portion and at the periphery of the antimesometrial portion of the decidua at Embryonic Day (E) 8.5, and in the parietal endoderm surrounding the embryo. Serglycin mRNA was detected in fetal liver at E11.5-E14.5. Serglycin was detected by immunohistochemistry in decidua and parietal endoderm at E8.5 and in liver at E13.5. Most of the proteoglycans synthesized by cultured intact deciduae (78%) and adherent decidual cultures (91%) were secreted into the medium. Serglycin proteoglycan may play an important role in uterine decidual function during early postimplantation development.

Subtractive hybridization reveals tissue-specific expression of ahnak during embryonic development.

The gene product ahnak has been identified from extra-embryonic mesoderm cDNA enriched using a subtractive hybridization approach modified for using small amounts of starting material. Clones for cyclin D2 and H19 have also been isolated as being preferentially enriched in the extra-embryonic mesoderm compared with the embryo proper of embryonic day (E) 7.5 neural plate stage mouse embryos. The differential expression of these genes was confirmed at gastrulation stage using in situ hybridization. More detailed analysis of the human genomic ahnak sequence suggests that its highly repetitive structure was formed by unequal cross-over and gene conversion. During organogenesis, ahnak is expressed in a variety of tissues, including migratory mesenchyme. By E12.5, the major site of expression of ahnak is craniofacial mesenchyme. Immunohistochemical analysis has shown that ahnak protein is expressed mainly at the cell membrane of migratory mesenchymal cells, primarily in the nucleus of bone growth plate cells and mostly in the cytoplasm of differentiating nasal epithelia. The potential functions of ahnak are discussed in light of these results.

Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4.

Directed cell movement is integral to both embryogenesis and hematopoiesis. In the adult, the chemokine family of secreted proteins signals migration of hematopoietic cells through G-coupled chemokine receptors. We detected embryonic expression of chemokine receptor messages by RT-PCR with degenerate primers at embryonic day 7.5 (E7.5) or by RNase protection analyses of E8.5 and E12.5 tissues. In all samples, the message encoding CXCR4 was the predominate chemokine receptor detected, particularly at earlier times (E7.5 and E8.5). Other chemokine receptor messages (CCR1, CCR4, CCR5, CCR2, and CXCR2) were found in E12.5 tissues concordant temporally and spatially with definitive (adult-like) hematopoiesis. Expression of CXCR4 was compared with that of its only known ligand, stromal cell-derived factor-1 (SDF-1), by in situ hybridization. During organogenesis, these genes have dynamic and complementary expression patterns particularly in the developing neuronal, cardiac, vascular, hematopoietic, and craniofacial systems. Defects in the first four of these systems have been reported in CXCR4- and SDF-1-deficient mice. Our studies suggest new potential mechanisms for some of these defects as well as additional roles beyond the scope of the reported abnormalities. Earlier in development, expression of these genes correlates with migration during gastrulation. Migrating cells (mesoderm and definitive endoderm) contain CXCR4 message while embryonic ectoderm cells express SDF-1. Functional SDF-1 signaling in midgastrula cells as well as E12.5 hematopoietic progenitors was demonstrated by migration assays. Migration occurred with an optimum dose similar to that found for adult hematopoietic cells and was dependent on the presence of SDF-1 in a gradient. This work suggests roles for chemokine signaling in multiple embryogenic events.

Expression of homeobox genes, including an insulin promoting factor, in the murine yolk sac at the time of hematopoietic initiation.

The visceral yolk sac (YS), a simple bilayer structure formed during gastrulation, supplies blood cells and intestine- and liver-like functions to support embryonic growth. To better understand gene regulation in extraembryonic tissues, we examined the early murine YS for expression of the homeobox family of developmental transcription regulators. We identified a subset of known homeobox sequences (Hox 1l, b1, a9, c9, a7, b7, b8, a10, cdx-1, and PDX-1), as well as two novel homeodomains consisting of a fourth labial class Hox genes and one that matches the Antennapedia class on the amino acid level. The two most frequently isolated YS Hox genes, a9 and c9, are initially expressed only in the YS (E.5) and subsequently expressed in both the embryo and YS (E8.5). Another of the identified genes, PDX-1, is involved in pancreatic development and insulin regulation. Whereas the4 rodent YS is known to produce insulin from mid to late gestation, YS insulin expression had not been examined earlier in development . We detected insulin mRNA in the YS at both E7.5 and E8.5, prior to expression in the embryo proper or formation of the pancreas. However, other pancreatic products, such as glucagon, somatostatin, and carboxypeptidase A, are not expressed in the YS. In situ analysis indicates insulin is produced in YS mesothelial cells and endoderm cells, but not in blood cells. We hypothesize the early expression of insulin in the YS is required for the expansion of insulin responsive cells including primitive erythroblasts.

An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

Convergent control of serotonin transporter expression by glucocorticoids and cocaine in fetal and neonatal rat brain.

Serotonin plays a trophic role in brain cell differentiation. In this study, expression of the serotonin presynaptic transporter protein, which regulates the extracellular serotonin concentration, was measured with [3H]paroxetine in rats exposed to dexamethasone or cocaine prenatally. Within 24 h of a single dose of dexamethasone, significant increases were seen in fetal brain, and the effect persisted into the postnatal period. Chronic prenatal cocaine exposure elicited similar changes. These data indicate that exposures to apparently disparate drugs can elicit similar endpoints that may lead to behavioral teratogenesis.

Initiation of hematopoiesis and vasculogenesis in murine yolk sac explants.

The blood islands of the visceral yolk sac (VYS) are the initial sites of hematopoiesis in mammals. We have developed a yolk sac explant culture system to study the process of blood cell and endothelial cell development from extraembryonic mesoderm cells. No benzidine-positive cells or beta H1-globin mRNA expression was detected at the primitive streak or neural plate stage of development (E7.5). However, when isolated E7.5 dissected tissues were cultured for 36 to 72 hours in serum-free medium, hundreds of hemoglobin-producing cells and embryonic globin gene expression were identified in both intact yolk sac and VYS mesoderm explants. Explanted E7.5 extraembryonic mesoderm tissues thus recapitulate in vivo primitive erythropoiesis and do not require the presence of a vascular network or the VYS endoderm. Yolk sac blood islands also contain endothelial cells that arise by vasculogenesis and express flk-1. We detected flk-1 mRNA as early as the primitive streak stage of mouse embryogenesis. Culture of embryo proper and intact VYS explants, which contain both mesoderm and endoderm cells, produced capillary networks and expressed flk-1. In contrast, vascular networks were not seen when VYS mesoderm was cultured alone, although flk-1 expression was similar to that of intact VYS explants. The addition of vascular endothelial growth factor to VYS mesoderm explants did not induce vascular network formation. These results suggest that the VYS endoderm or its extracellular matrix is necessary for the coalescence of developing endothelial cells into capillary networks.

Regulation and evolution of the single alpha-tubulin gene of the ciliate Tetrahymena thermophila.

The single alpha-tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha-tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha-tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha-tubulins. The single alpha-tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha-tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha-tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha-tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha-tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha-tubulin message amounts via message decay. However the complex control of alpha-tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates.

Perspectives on tubulin isotype function and evolution based on the observation that Tetrahymena thermophila microtubules contain a single alpha- and beta-tubulin.

We have cloned and sequenced the two beta-tubulin genes of the ciliated protozoan Tetrahymena thermophila. The two genes encode identical 443 amino acid peptides which are 99.7% identical to the beta-tubulin proteins of T. pyriformis and 95% identical to human beta 1 tubulin. T. thermophila contains only one alpha-tubulin gene (Callahan et al., 1984: Cell 36:441-445). Thus, all of the extremely diverse microtubule structures in this unicellular organism can be formed from a single alpha- and a single beta-tubulin peptide. We have also carried out a phylogenetic analysis of 84 complete beta-tubulin peptide sequences. This analysis supports two hypotheses regarding beta-tubulin evolution and function: 1) Multifunctional beta-tubulins are under greater evolutionary constraint than beta-tubulins present in specialized cells or in cells with very few microtubule related functions, which can evolve rapidly; and 2) Cells which form axonemes maintain a homogeneous population of tubulins.