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Biomedical subjects

M L Bloom

Publications and source records attributed to M L Bloom.

At least 19 recordsLinked to original sources
PubMed1999

Endothelial progenitor cells as putative targets for angiostatin.

Angiostatin, a product of the proteolytic cleavage of plasminogen, possesses potent antitumor and antiangiogenic properties in vivo. Studies with cultured endothelial cells suggest that under certain conditions, angiostatin inhibits the migration and proliferation of these cells or, alternatively, increases their rate of apoptosis. In general, the effects of angiostatin have been considerably less potent in vitro than in vivo. One potential explanation for this disparity is that the in vivo target of angiostatin is not the mature endothelial cell. Recently, evidence has accumulated to show that circulating endothelial progenitor cells (EPCs) contribute to neovascularization. In this study, we have isolated EPCs from human subjects and demonstrated that, in contrast to that of mature endothelial cells, the growth of EPCs is exquisitely sensitive to angiostatin. These results suggest that angiostatin and related compounds may exert their biological effects by inhibiting the contribution of EPCs to angiogenesis and not by altering the growth of mature endothelial cells.

Angiostatins↗
PubMed1999

Restoration of lymphoid populations in a murine model of X-linked severe combined immunodeficiency by a gene-therapy approach.

X-linked severe combined immunodeficiency (XSCID) is a life-threatening syndrome in which both cellular and humoral immunity are profoundly compromised. This disease results from mutations in the IL2RG gene, which encodes the common cytokine receptor gamma chain, gamma(c). Previously, we generated gamma(c)-deficient mice as a murine model of XSCID. We have now used lethally irradiated gamma(c)-deficient mice to evaluate a gene therapeutic approach for treatment of this disease. Transfer of the human gamma(c) gene to repopulating hematopoietic stem cells using an ecotropic retrovirus resulted in an increase in T cells, B cells, natural killer (NK) cells, and intestinal intraepithelial lymphocytes, as well as normalization of the CD4:CD8 T-cell ratio and of serum Ig levels. In addition, the restored cells could proliferate in response to interleukin-2 (IL-2). Thus, our results provide added support that gene therapy is a feasible therapeutic strategy for XSCID. Moreover, because we used a vector directing expression of human gamma(c) to correct a defect in gamma(c)-deficient mice, these data also indicate that human gamma(c) can cooperate with the distinctive cytokine receptor chains such as IL-2Rbeta and IL-7Ralpha to mediate responses to murine cytokines in vivo.

Animals↗
PubMed1999

The hemoglobin-deficit mouse: cure of the anemia following bone marrow transplantation with normal marrow.

The hemoglobin-deficit mouse mutant (hbd) is characterized by a severe microcytic anemia that is inherited in an autosomal-recessive manner. Previous results from our laboratory indicated that normal mice develop anemia if they are transplanted with bone marrow from mutant animals. Furthermore, we demonstrated a delay in erythroid reconstitution from hbd marrow compared to normal marrow. Although these experiments show a defect that is intrinsic to hbd marrow, it is unclear if the hbd phenotype is solely the result of a bone marrow-derived defect. To exclude an environmental influence on hbd anemia, we attempted to cure the defect by transplanting normal marrow into the hbd mouse. We observed that the transplanted animals converted to a normal phenotype. These results indicated that the defect is bone marrow derived. In contrast to the microcytosis mutant whose defective gene is ubiquitously expressed, our data suggest that the defective gene product is specific to hematopoietic cells.

Anemia↗
PubMed1999

Retrovirus integration site Mintb encoding the mouse homolog of hnRNP U.

Retroviral genes are not usually expressed in mouse embryonal carcinoma (EC) cells, but they are readily expressed upon differentiation of these cells. We previously reported the isolation of EC cell lines that express a neomycin resistance (neo) gene introduced by a recombinant transducing Moloney murine leukemia virus from specific integration sites, Minta, Mintb, Mintc, or Mintd. In some of these clones, the entire 5' long terminal repeat (LTR) was deleted, and the neo gene was expressed by read-through transcription from upstream cellular promoters in a "promoter-trap" fashion. One such promoter ("promoter B" at the Mintb locus) was found in a CpG island, associated with an upstream enhancer ("enhancer B"). Although enhancer B caused expression of the neo gene in the transductant EC cell line, no endogenous transcription from promoter B was detected in the parental EC or NIH3T3 cells. In contrast, we found a strong counter-flow endogenous transcription unit ("R" for reverse), which apparently interfered with transcription from promoter B. Promoter R turned out to have a bidirectional activity in transfection assays. In normal tissues, promoter R activates gene R, which encodes an 800-residue protein that is highly homologous to the rat and human heterogeneous nuclear ribonucleoprotein U (hnRNP U). Northern and in situ hybridization analyses revealed that gene R was abundantly expressed in the testis, especially in the pachytene spermatocytes and round spermatids.

3T3 Cells↗
PubMed1998

Murine bone marrow expressing the neomycin resistance gene has no competitive disadvantage assessed in vivo.

The neomycin phosphotransferase (neo) gene is one of the most common marker genes used in gene transfer experimentation, but potential effects of neo gene expression in vivo have not been systematically investigated. Several early clinical retroviral gene transfer studies have suggested that neo gene expression could have deleterious effects on hematopoiesis, owing to a discrepancy between the level of neo-marked transduced marrow progenitor cells compared with mature circulating progeny cells posttransplantation (Brenner et al., 1993; Kohn et al., 1995; Brenner, 1996b). We examined the long-term in vivo repopulating ability of bone marrow from transgenic mice expressing neo from a strong constitutive promoter using a competitive repopulation assay. Different ratios of neo transgenic and wild-type congenic marrow cells were cotransplanted into W/Wv recipient mice. The percentages of blood cells containing the neo transgene in each group of recipient mice monitored for 4 months posttransplantation closely matched the input ratios of neo transgenic to congenic control marrow cells. Similar concordances of engraftment with input ratios of neo transgenic cells were also found in spleen, thymus, and whole marrow of recipient mice at 4 months posttransplantation. Analysis of the beta-hemoglobin phenotype (beta(single) for the neo transgenic and C57 control cells and beta(diffuse) for the congenic competitor HW80 cells) in recipients confirmed erythroid repopulation from neo transgenic marrow cells at levels matching the input ratios. We conclude that hematopoietic cells expressing neo had no engraftment or maturation defects detectable in vivo. These results suggest that the low-level contribution of vector-marked cells to circulating populations in clinical trials is not due to direct deleterious effects of neo gene expression on hematopoiesis.

Animals↗
PubMed1997

The hemoglobin-deficit mouse: analysis of phenotype and hematopoiesis in the transplant model.

The mouse mutant hemoglobin deficit (gene symbol hbd) is characterized by a severe microcytic anemia that is inherited in an autosomal-recessive manner. To assess the mutation's effect on hematopoiesis, unfractionated bone marrow (BM) from either a mutant C57BL6/J-hbd/hbd, Gpi1b/Gpi1b (phenotype symbol HBD), or normal C57BL6/J-+hbd/+hbd, Gpi1b/Gpi1b mouse was injected intravenously into irradiated congenic C57BL6/J-+hbd/+hbd, Gpi1a/Gpi1a, lgh(a)/lgh(a), Thy1a/Thy1a mice. The congenic recipients of mutant or normal marrow obtained complete red blood cell (RBC) and leukocyte reconstitution, with the exception of one recipient of HBD marrow. After 24 weeks posttransplantation, the normal recipients of HBD marrow obtained a microcytic anemia similar to the donor. These results suggest that the HBD phenotype is caused by a BM defect. We observed that the erythroid lineage derived from donor HBD marrow repopulated more slowly than the normal marrow at 4 weeks post-transplantation. To determine if this difference was a result of an erythropoietic defect, competitive repopulation was performed using either mutant or normal marrow competed against normal congenic marrow. For the erythroid lineage, no significant contribution from HBD marrow was observed. To assess if the RBC block was based on a deficiency of myeloid progenitors, both in vitro and in vivo assays were performed: absolute numbers of bone progenitors were increased, suggesting that the defect results in a late block to erythroid differentiation.

Animals↗
PubMed1995

Brain spectrin: of mice and men.

This article reviews our current knowledge of the structure of alpha spectrins and beta spectrins in the brain, as well as their location and expression within neural tissue. We discuss the known protein interactions of brain spectrin isoforms, and then describe results that suggest an important role for spectrin (alpha SpII sigma 1/beta SpII sigma 1) in the Ca(2+)-regulated release of neurotransmitters. Evidence that supports a role for spectrin in the docking of synaptic vesicles to the presynaptic plasma membrane and as a Ca2+ sensor protein that unclamps the fusion machinery is described, along with the Casting the Line model, which summarizes the information. We finish with a discussion of the value of spectrin and ankyrin-deficient mouse models in deciphering spectrin function in neural tissue.

Amino Acid Sequence↗
PubMed1994

Brain alpha erythroid spectrin: identification, compartmentalization, and beta spectrin associations.

Using isoform and subunit specific antibodies we have determined the presence, localization, and beta spectrin associations of alpha erythroid spectrin, alpha SpI sigma*, as well as alpha non-erythroid spectrin, alpha SpII sigma 1, in mouse brain. Peptide specific antibodies against unique sequences within the beta SpII sigma 1, non-erythroid beta spectrin isoform, and within beta SpI sigma 1, erythrocyte beta spectrin isoform were used to compare the immunolocalization of beta spectrin subunit isoforms with that of alpha spectrin subunit isoforms and to immunoprecipitate spectrin tetramers in order to identify the subunit components by immunoblot analysis. The specificity and sensitivity of antibodies for isoform specific alpha and beta subunits was determined by immunodot and immunoblot methods. Immunohistochemical analyses indicated that beta SpI sigma 2 is located in neuronal somata and dendrites in mouse cerebellum. beta SpII sigma 1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemical analysis with antibodies for the alpha and beta spectrin isoforms revealed that antibodies specific for the alpha subunit of erythrocyte spectrin (alpha SpI sigma 1) localized antigen to the somata and dendrites of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid beta subunit (beta SpI sigma 2). In contrast antibodies specific for the non-erythroid alpha subunit (alpha SpII sigma 1) localized antigen to axons in the cerebellum corresponding to the pattern for the non-erythroid beta subunit (beta SpII sigma 1). The distinct localization of antigens by antisera which recognize either the alpha subunit of red blood cell spectrin or the alpha subunit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate beta subunits, clearly indicate that brain contains at least two species of spectrin each with distinct alpha and beta subunits. Immunoprecipitation experiments of cerebellar extracts using beta spectrin peptide specific antibodies followed by immunoblotting analysis confirmed the association of an erythroid alpha subunit isoform with a beta erythroid subunit isoform, as well as the association of non-erythroid alpha and beta subunits. In addition the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain consisting of mixed erythroid and non-erythroid subunits. In summary these data collectively demonstrate that in mouse brain there are at least two alpha spectrin subunits, one erythroid alpha SpI sigma* and one non-erythroid alpha SpII sigma 1; these associate with an erythroid beta SpI sigma 1, and a non-erythroid beta SpII sigma 1 in the cerebellum of mouse.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

The murine mutation jaundiced is caused by replacement of an arginine with a stop codon in the mRNA encoding the ninth repeat of beta-spectrin.

The jaundiced, ja/ja, mouse mutant has a severe hemolytic anemia associated with a deficiency of beta-spectrin in erythrocyte ghosts. Genes for the disease phenotype and beta-spectrin colocalize on Chromosome 12. beta-Spectrin mRNA is not detected in reticulocytes or in brain from newborn mutant mice. To locate the nucleotide sequence alteration, the erythroid beta-spectrin transcript from mutant spleen was amplified by reverse transcription PCR and sequenced. A C-to-T alteration is present in the mutant transcript and produces a premature stop codon from an arginine codon in mRNA encoding repeat 9 of beta-spectrin at amino acid position 1160. The point mutation introduces a Dde I site that is present in PCR-amplified DNA of ja/ja and ja/+ mice but not of +/+ control mice from the strain of origin, 129/Sv, or from the two strains, WB/Re and C57BL/6J, in which the mutation has been fixed by over 53 generations of backcrossing. The genetic data confirm that the point mutation is responsible for the severe reductions in beta-spectrin mRNA of jaundiced mice.

Amino Acid Sequence↗
PubMed1993

Complete nucleotide sequence of the murine erythroid beta-spectrin cDNA and tissue-specific expression in normal and jaundiced mice.

Spectrin, a heterodimer of alpha and beta subunits, is an essential component of the red blood cell membrane skeleton. The jaundiced (ja/ja) mutation causes a severe hemolytic anemia in mice and is mapped to the erythroid beta-spectrin locus (Spnb-1) on chromosome 12. As a prerequisite for determining the molecular defect of the jaundiced mutation, we have cloned and sequenced the complete murine reticulocyte cDNA for normal Spnb-1. Two unique transcripts that differ in the placement of polyA tails are represented in the clones isolated. Amino acid sequence comparison between erythroid and murine brain spectrin (Spnb-2, chromosome 11) shows 67% identity throughout repeats 16 and 17 and complete divergence in domain III, which is associated with the alpha/beta subunit dimerization and phosphorylation. We examined the tissue distribution of normal and mutant erythroid beta-spectrin transcripts using domain-specific probes. Transcripts are detected in normal spleen tissue and reticulocytes (8 and 9.6 kb), brain tissue (10 and 11 kb), skeletal muscle tissue, and cardiac muscle tissue (11, 10.3, 7.2, and 4.0 kb). Extensive variability in mRNA processing is shown with region-specific probes. Steady state levels of the mutant transcripts are reduced when hybridized with a probe to repeats 2 through 6 with the exception of the 7.2-kb transcript that is unique to heart and skeletal muscle tissues, and is present at normal and elevated levels, respectively, in ja/ja mice. These results provide evidence for more diverse tissue-specific products of the Spnb-1 gene than were previously suspected.

Amino Acid Sequence↗
PubMed1993

The complete amino acid sequence for brain beta spectrin (beta fodrin): relationship to globin sequences.

The amino acid sequence of mouse brain beta spectrin (beta fodrin), deduced from the nucleotide sequence of complementary DNA clones, reveals that this non-erythroid beta spectrin comprises 2363 residues, with a molecular weight of 274,449 Da. Brain beta spectrin contains three structural domains and we suggest the position of several functional domains including f-actin, synapsin I, ankyrin and spectrin self association sites. Analysis of deduced amino acid sequences indicated striking homology and similar structural characteristics of brain beta spectrin repeats beta 11 and beta 12 to globins. In vitro analysis has demonstrated that heme is capable of specific attachment to brain spectrin, suggesting possible new functions in electron transfer, oxygen binding, nitric oxide binding or heme scavenging.

Actins↗
PubMed1992

Changing patterns in cytoskeletal mRNA expression and protein synthesis during murine erythropoiesis in vivo.

The major cytoskeletal proteins alpha-spectrin, beta-spectrin, and ankyrin are synthesized and assembled into a supportive membrane skeleton during erythroid differentiation. Information on the temporal appearance of mRNA and protein species is essential for understanding both the cytoskeletal assembly process and the function of various isoforms. We have isolated highly enriched populations of fetal erythroid cells at various stages of maturation. mRNAs for erythroid ankyrin, alpha-spectrin, and beta-spectrin were expressed at all stages but there were differences in transcript types and levels. The ratio of 9-kilobase (kb) to 7.5-kb erythroid ankyrin transcripts decreased markedly during differentiation, but there was no change in the ratio of the 10.1-kb and 9.3-kb erythroid beta-spectrin transcripts. The relative amounts of ankyrin, alpha-spectrin, and beta-spectrin mRNA increased during yolk sac cell differentiation, whereas only alpha-spectrin mRNA increased during differentiation of the fetal liver cells. The amounts of beta-spectrin mRNA exceeded the amounts of alpha-spectrin mRNA in the early precursors from both yolk sac and fetal liver; protein synthetic levels showed the same pattern. The 16-day fetal peripheral reticulocytes, on the other hand, had the adult mRNA and protein synthetic ratios with alpha/beta greater than 1. The data indicate that at least two mechanisms exist to meet changing erythroid membrane cytoskeletal requirements during development in utero: (i) stage-specific processing of the mRNA for the major cytoskeletal linker protein ankyrin and (ii) developmentally regulated alpha/beta-spectrin protein synthetic rates.

Animals↗
PubMed1985

Slipped capital femoral epiphysis. An assessment of treatment modalities.

Fifty-five patients underwent operative treatment of slipped capital femoral epiphyses at Children's Hospital Medical Center in Cincinnati, Ohio, between January 1975 and April 1980. Charts and x-rays were available for review in 45 of these patients with 68 affected hips; the incidence of bilaterality was 51%. Sixty-five of the 68 hips were managed by pinning. Problems with pin placement were recognized in 15.4% of these hips. Three patients were treated by subtrochanteric osteotomy, without complications. Since May 1980, all patients treated on the University Service, Children's Hospital Medical Center for slipped capital femoral epiphysis have undergone open epiphysiodesis utilizing a cosmetic "bikini" incision. Twenty-two hips in 14 patients were so managed without significant complications. This should be considered the treatment of choice for slips involving change greater than 30 degrees in the head shaft angle.

Child↗