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Biomedical subjects

A Schweitzer

Publications and source records attributed to A Schweitzer.

At least 19 recordsLinked to original sources
PubMed2006

Myelin basic protein functions as a microtubule stabilizing protein in differentiated oligodendrocytes.

Myelin basic protein (MBP) is an oligodendrocyte-specific protein essential for oligodendrocyte morphogenesis at late stages of cell differentiation. There is evidence that the morphogenetic function of MBP is mediated by MBP interaction with the cytoskeleton. Thus, an MBP/cytoplasmic microtubule association has been reported, and MBP has Ca(2+)/calmodulin-regulated microtubule cold-stabilizing activity in vitro. However, the unambiguous demonstration of a microtubule-stabilizing activity for MBP in cells has been difficult because oligodendrocytes contain variants of STOP (stable tubule only polypeptide) proteins, which are responsible for microtubule cold stability in different cell types. Herein, we have used genetic mouse models and RNA interference to assay independently the microtubule cold-stabilizing activities of MBP and of STOP in developing oligodendrocytes. In wild-type oligodendrocytes, microtubules were cold stable throughout maturation, which is consistent with the presence of STOP proteins from early stages of differentiation. In contrast, in oligodendrocytes from STOP-deficient mice, microtubules were cold labile in the absence of MBP expression or when MBP expression was restricted to the cell body and became stable in fully differentiated oligodendrocytes, where MBP is expressed in cell extensions. The suppression of MBP by RNA interference in STOP-deficient oligodendrocytes suppressed microtubule cold stability. Additionally, STOP suppression in oligodendrocytes derived from shiverer mice that lack MBP led to the complete suppression of microtubule cold stability at all stages of cell differentiation. These results demonstrate that both STOP and MBP function as microtubule-stabilizing proteins in differentiating oligodendrocytes and could be important for the morphogenetic function of MBP.

Animals↗
PubMed2004

Astrocytes and oligodendrocytes express different STOP protein isoforms.

Many cell types contain subpopulations of microtubules that resist depolymerizing conditions, such as exposure to cold or to the drug nocodazole. This stabilization is due mainly to polymer association with STOP proteins. In mouse, neurons express two major variants of these proteins, N-STOP and E-STOP (120 kDa and 79 kDa, respectively), whereas fibroblasts express F-STOP (42 kDa) and two minor variants of 48 and 89 kDa. N- and E-STOP induce microtubule resistance to both cold and nocodazole exposure, whereas F-STOP confers microtubule stability only to the cold. Here, we investigated the expression of STOP proteins in oligodendrocytes and astrocytes in culture. We found that STOP proteins were expressed in precursor cells, in immature and mature oligodendrocytes, and in astrocytes. We found that oligodendrocytes express a major STOP variant of 89 kDa, which we called O-STOP, and two minor variants of 42 and 48 kDa. The STOP variants expressed by oligodendrocytes induce microtubule resistance to the cold and to nocodazole. For astrocytes, we found the expression of two STOP variants of 42 and 48 kDa and a new STOP isoform of 60 kDa, which we called A-STOP. The STOP variants expressed by astrocytes induce microtubule resistance to the cold but not to nocodazole, as fibroblast variants. In conclusion, astrocytes and oligodendrocytes express different isoforms of STOP protein, which show different microtubule-stabilizing capacities.

Animals↗
PubMed2001

Identification of novel bifunctional calmodulin-binding and microtubule-stabilizing motifs in STOP proteins.

Although microtubules are intrinsically labile tubulin assemblies, many cell types contain stable polymers, resisting depolymerizing conditions such as exposure to the cold or the drug nocodazole. This microtubule stabilization is largely due to polymer association with STOP proteins. There are several STOP variants, some with capacity to induce microtubule resistance to both the cold and nocodazole, others with microtubule cold stabilizing activity only. These microtubule-stabilizing effects of STOP proteins are inhibited by calmodulin and we now demonstrate that they are determined by two distinct kinds of repeated modular sequences (Mn and Mc), both containing a calmodulin-binding peptide, but displaying different microtubule stabilizing activities. Mn modules induce microtubule resistance to both the cold and nocodazole when expressed in cells. Mc modules, which correspond to the STOP central repeats, have microtubule cold stabilizing activity only. Mouse neuronal STOPs, which induce both cold and drug resistance in cellular microtubules, contain three Mn modules and four Mc modules. Compared with neuronal STOPs, the non-neuronal F-STOP lacks multiple Mn modules and this corresponds with an inability to induce nocodazole resistance. STOP modules represent novel bifunctional calmodulin-binding and microtubule-stabilizing sequences that may be essential for the generation of the different patterns of microtubule stabilization observed in cells.

Amino Acid Motifs↗
PubMed2001

[Electrophysiologic study of 10 cases of Miller Fisher syndrome].

The association of ophthalmoplegia, ataxia and areflexia was described by Miller Fisher in 1956. It is postulated as a variant of the Guillain Barré syndrome. We report 10 Miller Fisher syndrome patients admitted in an intensive care unit between June 1990 and February 1999 who were selected according to clinical criteria of Ropper and Wijdicks. All patients had motor and sensory nerve conduction studies and electromyography, nine had visual and brainstem auditory evoked potentials and two had short latency somatosensory evoked potentials. Peripheral neuropathy was found in all patients. All had sensory nerve changes and some were severe. Motor nerve conduction abnormalities were observed in 7 only cases with moderate increase of F latency in 3 cases and compound muscle action potential reduction in 3 other cases. In the last case, motor conduction abnormalities was more severe, characterized by conduction velocity slowing in both distal and proximal sites and by temporal dispersion of action potentials. All brainstem auditory evoked studies were normal. In 4 patients, MRI studies were normal. These data support that brainstem is preserved in MFS. Only one patient had visual evoked potential abnormalities. Optic neuropathy is debated in Miller Fisher and in Guillain Barré syndrome. As a conclusion, in MFS peripheral neuropathy is always present with severe sensitive changes and moderate motor changes (This is different as compared to Guillain Barré syndrome according to electrophysiological data). We did not find involvement of brainstem in our patients with Miller Fisher syndrome.

Adolescent↗
PubMed1999

The journey to multiple sclerosis: a qualitative study.

The diagnosis of multiple sclerosis (MS) often creates more questions than answers. It is not uncommon to hear about individuals who receive the diagnosis with relief and surprise, However, the implications of being diagnosed with a chronic illness like MS is complex. There is no known cure for this disease that has remissions, relapses and possible continued progression. For individuals diagnosed with MS, responding to the physical and psychological stresses of a condition with an unknown cause and no cure is challenging. The purpose of this study was to explore the individual's experience of having symptoms for a period of time and then to be told they have MS. The phenomenological qualitative research method was used to illuminate the nature of their experiences. The time course spanned before diagnosis, at diagnosis and following diagnosis. Interviews were conducted with five purposefully selected participants. The process of data analysis highlighted common threads and patterns among informants. Four major themes emerged: Whispered Beginnings, Echoes of Silence, The Spoken Words, and Recreating Voice. The experiences of individuals recently diagnosed with MS are illuminated in order to provide insight and information to health care providers who are faced with telling a person the diagnosis of MS.

Activities of Daily Living↗
PubMed1998

Adhesion of mature polyploid megakaryocytes to fibronectin is mediated by beta 1 integrins and leads to cell damage.

Human CD34+ bone marrow cells were committed to the megakaryocytic lineage in serum-free liquid cultures by the following cytokines: thrombopoietin, erythropoietin, and IL-6. Megakaryocyte maturation has been described as being regulated by the extracellular matrix. These cells express receptors for laminin, collagen, and vitronectin, but they selectively adhere to and spread on fibronectin, a major component of the bone marrow environment. Function-perturbing antibodies against beta 1 integrins totally abolished the adhesion of megakaryocytes on fibronectin, whereas antibodies to beta 3 did not, suggesting that beta 1 integrins were responsible for the adhesive phenotype of these polyploid cells. beta 1-positive clusters were visualized in close contact with the extremities of stress fibers at the cell surface. In the course of cell spreading, we observed morphological modifications such as the disorganization of the compact nuclei structure and the appearance of holes in the cytoplasm leading to the release of alpha IIb beta 3-positive cellular fragments. This process appeared to be a specific feature of megakaryocytes and is correlated neither to apoptosis nor to integrin signaling.

Actin Cytoskeleton↗
PubMed1998

Voice recognition--an emerging necessity within radiology: experiences of the Massachusetts General Hospital.

Voice recognition represents a technology that is finally ready for prime time use. As radiology services continue to acquire a larger percentage of the shrinking health-care dollar, decreasing operating costs and improved services will become a necessity. The benefits of voice recognition implementation are significant, as are the challenges. This report will discuss the technology, experiences of major health-care institution with implementation, and potential benefits for the radiology practice.

Humans↗
PubMed1997

Targeted null-mutation in the vascular endothelial-cadherin gene impairs the organization of vascular-like structures in embryoid bodies.

Vascular endothelial-cadherin (VE-cadherin) is exclusively expressed in endothelial cells and is strictly located at cell-to-cell junctions. As the other members of the cadherin family, VE-cadherin is able to mediate a homotypic type of cellular interaction in a Ca2+-dependent manner. In the mouse embryo, VE-cadherin transcripts are detected at the earliest stages of vascular development. To ascertain if VE-cadherin expression is required for the assembly of endothelial cells into vascular structures, we generated VE-cadherin-negative mouse embryonic stem cells (VE-cadherin-/- ES cells) by gene targeting and examined the consequences on vascular development of ES-derived embryoid bodies (EBs). In contrast to wild-type EBs, we observed that endothelial cells remained dispersed and failed to organize a vessel-like pattern in VE-cadherin-/- ES-derived EBs. However, dispersed VE-cadherin-/- ES-derived endothelial cells expressed a large range of other endothelial markers. Moreover, the targeted null-mutation in the VE-cadherin locus did not interfere with the hematopoietic differentiation potential of ES cells. These in vitro experiments are consistent with a pivotal role of VE-cadherin in vascular structure assembly.

Animals↗
PubMed1997

Effects of retinoic acid on a new human erythromegakaryocytic cell line AP-217.

We have characterized a new human cell line AP-217, derived from the peripheral blood of a patient with chronic myeloid leukemia in blastic crisis. The analysis of cell surface antigens and ploidy showed that AP-217 was an erythro-megakaryocytic cell line. The effects of inducers of differentiation were studied and focused on retinoic acid (RA). Uninduced AP-217 cells produced a low level of hemoglobin (Hb) that showed a moderate but significant dose-dependent increase after 13 cis-RA induction (four times above the control at 10(-5) M). To outline this effect, AP-217 cells were cloned at limiting dilution. A subclone (clone 2) was isolated which expressed glycophorin A on 12% of cells, and showed a marked sensitivity to RA. After a 4 day induction with increasing concentrations of RA (1-10 x 10(-6) M) Hb production by clone 2 cells was enhanced 12 times over the control at the highest concentration (10(-5) M). No effect of RA on the Hb production of K-562 and HEL was observed. This increased Hb production occurred simultaneously with a growth inhibition in clonogenic cultures (20% reduction) associated with a drastic reduction of the colony size. Moreover, we demonstrated the expression of mRNA for the beta globin gene in clone 2 and AP-217-cells. This is the first report of a positive effect of RA on the erythroid differentiation of a human leukemic cell line.

Adult↗
PubMed1997

Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro.

The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not in the megakaryocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34+ purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA-2), fibronectin (VLA-4 and VLA-5) and laminin (VLA-6) during the maturation of Mks. We have shown that a significant percentage of CFU-MK (roughly 20%) adhere to fibronectin but not to collagen and laminin. The expression and adhesion of Mks developing in liquid culture from BM-CD34+ cells were tested at days 4, 7 and 10 of incubation. The expression of VLA-2, VLA-5 and VLA-6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FACS sorting whereas VLA-4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin. Our data suggest that VLA-4 may be involved in the adhesion of CFU-MK and immature Mks on fibronectin, then replaced by VLA-5 in the final stages of maturation. The expression of VLA-6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.

Antigens, CD34↗
PubMed1997

The MS-5 murine stromal cell line and hematopoietic growth factors synergize to support the megakaryocytic differentiation of embryonic stem cells.

Murine embryonic stem (ES) cells are able to differentiate into erythroid, mast, and granulomonocytic cells by using appropriate culture conditions. Because we were interested in the regulation of tissue-specific expression of the platelet glycoprotein IIb gene, we studied the culture conditions, aiming at the reproducible production of myeloid cells that included megakaryocytes (MKs) from ES cells. We showed that even a complex cocktail of HGFs (stem cell factor, interleukin 3, IL6, IL11, granulocyte colony-stimulating factor, and erythropoietin) is unable to induce significant myeloid differentiation in day 12 embryoid bodies. Cocultures of MS-5 stromal cells with ES cells were slightly more productive than HGFs. A strong synergistic effect was observed on the growth of myeloid colonies and MKs when we used a combination of MS-5 cells plus the HGF cocktail. Conditioned medium from MS-5 cells also synergized with the HGF cocktail to produce a substantial number of mixed colonies containing MKs. The addition of fibroblast growth factor-2 (FGF-2) to the HGF cocktail plus MS-5 nearly doubled the number of myeloid progenitors, including those with MKs. Thrombopoietin (TPO) alone or in any combination with MS-5 or HGFs, did not increase the number of MK-containing colonies. However, when TPO was added to the HGF cocktail + FGF-2 + MS-5, the number of MKs in liquid cultures and mixed colonies increased, and many exhibited a "hairy" appearance resembling pseudopodial proplatelet formation. Having defined the culture conditions of ES cells that allow the production of all the myeloid lineages including MKs, we conclude that the hematopoietic differentiation model of ES cells is especially useful for studying the regulation of expression of any gene important in early hematopoiesis.

Animals↗
PubMed1996

Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps.

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.

Animals↗
PubMed1995

Beta 1 integrins mediate adherent phenotype of human erythroblastic cell lines after phorbol 12-myristate 13-acetate induction.

We investigated the effects of phorbol ester (phorbol 12-myristate 13-acetate; PMA) treatment on the adhesive behaviour of three erythroleukaemia cell lines: HEL, LAMA-84 and AP217. In the three cell lines PMA induced an increase in expression of a megakaryocytic marker: alpha IIb beta 3 integrin, but did not promote activation of this receptor. Indeed, an antibody specific for the activated form of alpha IIb beta 3 failed to react with the three cell lines. PMA induction led to different adhesive phenotypes depending on the cell line; in fact LAMA-84 and HEL cells became adherent while AP217 cells remained non-adherent. By studying cell surface receptors we found that the major difference between the adherent and the non-adherent cells was the expression of beta 1 integrins. After PMA induction, beta 1 integrin expression was totally abolished in AP217 cells and the amount of beta 1 mRNA was reduced preventing new synthesis of the subunit. In HEL and LAMA-84 cells, PMA treatment did not alter the overall level of beta 1 integrin but induced a new pattern of alpha-subunit expression: up-regulation of alpha 2 and alpha v subunits and down-regulation of alpha 4 and alpha 5 subunits. Function-perturbing antibodies against beta 1, alpha 4, alpha 5, alpha v and alpha 2 reduced adhesion of HEL cells to fibronectin or collagen, whereas antibodies against beta 3 or alpha v beta 3 did not. Our results favour the involvement of beta 1 integrins in PMA-induced adhesion of erythroleukaemia cells.

Base Sequence↗
PubMed1995

Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein alpha IIb promoter.

The mechanisms that regulate the commitment of a totipotent stem cell to the megakaryocytic lineage are largely unknown. Using a molecular approach to the study of megakaryocytopoiesis and platelet production, mice in which thrombocytopoiesis could be controlled were produced by targeting the expression of the herpes simplex virus thymidine kinase toxigene to megakaryocytes using the regulatory region of the gene encoding the alpha subunit of the platelet integrin alpha IIb beta 3. The programmed eradication of the megakaryocytic lineage was induced by treating transgenic mice bearing the hybrid construct (alpha IIbtk) with the antiherpetic drug ganciclovir (GCV). After 10 d of treatment, the platelet number was reduced by > 94.6%. After discontinuing GCV, the bone marrow was repopulated with megakaryocytes and the platelet count was restored within 7 d. Prolonged GCV treatment induced erythropenia in the transgenic mice. Assays of myeloid progenitor cells in vitro demonstrated that the transgene was expressed in early erythro-megakaryocytic progenitor cells. The reversibility and facility of this system provides a powerful model to determine both the critical events in megakaryocytic and erythroid lineage development and for evaluating the precise role that platelets play in the pathogenesis of a number of vascular occlusive disorders.

Animals↗
PubMed1994

Evidence for a selective inhibitory effect of thrombin on megakaryocyte progenitor growth mediated by the thrombin receptor.

An efficient method for the culture of human megakaryocyte precursors in serum-free medium has been developed facilitating study of the effect of regulators of megakaryocyte growth and maturation without interference by serum-derived factors. We have investigated how megakaryocytes and their precursors respond to the procoagulant enzyme, thrombin. In addition to its already documented agonist effect on mature megakaryocytes, thrombin was found to have a marked inhibitory effect on the growth of megakaryocyte colonies from CD34+ bone marrow cells stimulated by IL3. This inhibitory effect, not previously reported, was selective for megakaryocytic cells. The growth of granulomonocytic and erythroid colonies was not affected. A monoclonal antibody which neutralized the effect of exogenous transforming growth factor beta (TGF beta) was unable to fully neutralize the inhibitory effect of thrombin. With the use of a synthetic peptide, corresponding to the tethered thrombin receptor ligand, and of a recombinant inactive form of thrombin, we provide direct evidence that both the inhibitory effect of thrombin on megakaryocyte proliferation and its agonist effect on mature megakaryocytes are mediated by a receptor analogous to the recently cloned platelet thrombin receptor.

Antigens, CD↗
PubMed1994

Detection of plasminogen activator inhibitor-1 (PAI-1) mRNA in human megakaryocytes by in situ hybridization.

Platelets have been described to contain a large proportion of the circulating plasminogen activator inhibitor type 1 (PAI-1) which is released on platelet activation. This protein could be taken up by platelets from the plasma or synthesized by megakaryocytes (MKs). Recently, PAI-1 mRNA has been detected in a human megakaryoblastic leukemia cell line (MEG-01) by the polymerase chain reaction (PCR). However, a direct-demonstration of its presence in normal human MKs is lacking. In order to prove directly the megakaryocytic origin of platelet PAI-1, the MEG-01 cell line, human bone marrow enriched in MKs, and bone marrow smears from allogeneic bone marrow transplantation donors were investigated for the presence of PAI-1 mRNA using in situ hybridization (ISH). Specimens of bone marrow were first stained with May-Grünwald Giemsa (MGG) for cell identification according to their morphology. Subsequently, the same slides were used for ISH. PAI-1 mRNA was clearly demonstrated in the MEG-01 cell line and in MKs, and its presence correlated with the detection of PAI-1 antigen by immunocytochemistry. PAI-1 mRNA was also detected in morphologically characterized mature granulocytes of marrow samples.

Bone Marrow↗
PubMed1993

Serum-free medium allows the optimal growth of human megakaryocyte progenitors compared with human plasma supplemented cultures: role of TGF beta.

The growth of human megakaryocyte progenitors from human bone marrow (BM) cells was compared using a methylcellulose semisolid assay supplemented either by normal human plasma or by a serum-free medium. Far better growth of megakaryocyte colonies from CD34+ BM cells stimulated by interleukin 3 (IL-3) and interleukin 6 (IL-6) was observed in serum-free medium compared with human plasma supplemented cultures. These results were confirmed in liquid cultures using the same serum-free medium composition. The megakaryocytes were identified by using an immunocytochemical procedure after labeling with an anti-GPIIb-IIIa monoclonal antibody. High percentages (15 to 20%) of megakaryocytes were present in serum-free cultures stimulated by IL-3 alone or combined with IL-6. The absolute number of megakaryocytes in serum-free medium exceeds by 3.3 (IL-3 plus IL-6) to 4.4 (IL-3 alone) times the corresponding number of megakaryocytes observed in human plasma supplemented cultures. The optimal concentration of IL-3 alone was 5 ng/ml, and an optimal synergistic effect of IL-6 (5 ng/ml) was obtained when combined with a suboptimal dose of IL-3 (1 ng/ml). The poor growth of megakaryocyte colonies from CD34+ BM cells in human plasma suggested the presence of an inhibitory factor. When a neutralizing monoclonal antibody against transforming growth factor beta (TGF beta) is present in human plasma supplemented cultures of CD34+ BM cells, the number of megakaryocyte colonies is increased to the level observed in corresponding serum-free cultures. The high efficiency of this serum-free medium to promote the growth of human megakaryocytes will be useful to study the effects of regulators and platelet agonists acting on human megakaryocytes, without interference from factors in the serum.

Antibodies, Monoclonal↗