Biomedical subjects

J J Minguell

Publications and source records attributed to J J Minguell.

At least 19 recordsLinked to original sources

PubMed1992

Expression of chondroitin sulfate as a unique type of proteoglycan on the cell membrane of multipotential and committed hemopoietic progenitor cells.

Proteoglycans are increasingly implicated as a major factor in the regulation of hemopoiesis. They are generally synthesized by stromal cells and released to the extracellular matrix. More recently the ability of hemopoietic progenitor cells to synthesize proteoglycans has come into focus. In the present study we maintained 3 cloned factor-dependent hemopoietic progenitor cells (B6 and F-mix which are multipotential and F-2 which is bipotential) in liquid culture. The cells were pulse-labeled with 35SO4 which becomes incorporated into the glycan side-chains of proteoglycans. We then studied subcellular distribution and chemical characterization of the newly synthesized proteoglycans. All 3 cell lines synthesized chondroitin sulfate as a unique type of proteoglycan as identified by gel filtration on a Sepharose CL-4B column followed by chondroitinase ABC cleavage of its glycosaminoglycan. This single type of proteoglycan was compartmentalized into intracellular, membrane-associated and extracellular pools. Its density on the membrane appeared to be a function of the differentiation state of the cell. The functional significance of membrane-associated proteoglycan in hemopoietic progenitor cells appears to be underestimated and requires further investigation.

Animals↗

PubMed1992

Collagen increases the synthesis of membrane-associated proteoglycans produced by Sertoli cells.

Sertoli cells in culture produce two isoforms of proteoglycans which are found in the culture medium and associated with the cell membrane. The amount of both types of proteoglycans increased when Sertoli cells were plated on type I collagen-coated dishes as compared to uncoated dishes. The effect is due to an increase in the synthesis of proteoglycans rather than a diminished rate of degradation of these molecules. The collagen substrate also affects the distribution of these macromolecules; an increase in the amount of membrane-associated proteoglycans occurs at the expense of the proteoglycans released to the culture medium.

Animals↗

PubMed1992

Membrane-associated chondroitin sulfate proteoglycan and fibronectin mediate the binding of hemopoietic progenitor cells to stromal cells.

The initial step in hemopoiesis is the binding of progenitor cells to stroma. What mediates this binding at the molecular level is not entirely clear. We have previously reported that the cell line FDCP-1, a factor-dependent hemopoietic progenitor cell, actively synthesizes a membrane-associated chondroitin sulfate (CS) proteoglycan (MA-PG) which is unstable. After the binding of the progenitor cell to stromal, the stability of the MA-PG is enhanced, suggesting its involvement in the binding of progenitor cells to the stroma. Since stromal cells possess pericellular fibronectin (FN), we examined the possibility that binding to stromal cells may involve interactions between MA-PG of FDCP-1 on the one side and pericellular FN in stromal cells on the other side. To examine this hypothesis, we developed a cell adherence assay to measure the binding of FDCP-1 cells to a monolayer of stromal cells or to FN-coated dishes. Cell binding was inhibited by a monoclonal antibody against CS as well as by free CS and heparin, suggesting the involvement of MA-PG in the binding. Pretreatment of FDCP-1 cells with chondroitinase ABC, which selectively removes the CS portion of the MA-PG, also affects binding to the stromal cells. The binding was also inhibited by a pentapeptide (GRGDS) which competes with the cell-binding domain of FN as well as by a monoclonal antibody anti-FN. We conclude that interactions between MA-PG and a putative integrin-like molecule in FDCP-1 and the heparin and the cell binding domains in pericellular FN in the stromal cells contribute to the stabilization of progenitor-stromal cell binding which originally comes about by homing receptors of progenitor cells.

Amino Acid Sequence↗

PubMed1992

Endothelial transcytosis of iron-transferrin in the liver does not involve endosomal traffic.

Through a process resembling receptor-mediated internalization, liver endothelium binds and internalizes iron-transferrin (Fe-Tf) complexes, transporting them from the luminal to abluminal side. Since in most systems, the path of receptor-mediated endocytosis leads to the endosomal compartment where the medium is acidified, it is expected that Fe and Tf become dissociated in this acidified medium. However, experiments with double labeling (59Fe, 125I-Tf) indicate that these remain associated. To determine whether the endosomal pathway is used in the course of transendothelial transport of Fe-Tf, experiments were done by incubating purified liver endothelium with radiolabeled Fe-Tf in the presence and absence of endosomal inhibitors, NH4Cl, ethylamine and monensin. The discharge of radiolabeled protein was measured as a function of time. While there was an early phase inhibition in the presence of endosomal inhibitors, the discharge of Tf by endothelium in the presence of inhibitors, reached a plateau comparable to control cells after 4 h, indicating that endosomal inhibition does not inhibit transendothelial transport of Tf and, thus, the transport does not involve endosomal traffic.

Ammonium Chloride↗

PubMed1992

Membrane-associated proteoglycans produced by Sertoli cells are not randomly distributed on the cell surface.

Sertoli cells in culture synthesize two membrane-associated proteoglycans (PGs) containing as glycosaminoglycan (GAG) moieties either chondroitin sulfate (CS) or CS and heparan sulfate (HS); the latter PG is, therefore, referred to as the mixed PG. To determine if these PGs are randomly distributed on the cell surface, Sertoli cell monolayers were treated with chondroitinase ABC, and then the remaining PGs were analyzed by DEAE-Sephacel chromatography. The results obtained with Sertoli cell monolayers show that the CS of the mixed PG is degraded by chondroitinase, while the CS-PG is not degraded. In contrast, chondroitinase treatment of Sertoli cells in suspension shows that both the mixed PG and the CS-PG are degraded. From this, it is inferred that the mixed PG is apically oriented and the CS-PG is basolaterally oriented. Studies of the adhesion of germ cells to Sertoli cell monolayers give further support to the apical location of the mixed PG and suggest that its HS moiety is involved in the attachment of germ cells to Sertoli cells.

Animals↗

PubMed1991

Enhancement of the grafting efficiency of transplanted marrow cells by preincubation with interleukin-3 and granulocyte-macrophage colony-stimulating factor.

To improve the grafting efficiency of transplanted murine hematopoietic progenitors, we briefly preincubated mouse bone marrow cells with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) ex vivo before their transplantation into irradiated recipients. This treatment was translated into an increase in the seeding efficiency of colony-forming unit-spleen (CFU-S) and CFU-GM after transplantation. Not only was the concentration of CFU-S in the tibia increased 2 and 24 hours after transplantation, but the total cell number and CFU-S and CFU-GM concentrations were persistently higher in IL-3- and GM-CSF-treated groups 1 to 3 weeks after transplantation. In addition, the survival of animals as a function of transplanted cell number was persistently higher in IL-3- and GM-CSF-treated groups compared with controls. The data indicate that the pretreatment of marrow cells with IL-3 and GM-CSF before transplantation increases the seeding efficiency of hematopoietic stem cells and probably other progenitor cells after transplantation. This increased efficiency may be mediated by upward modulation of homing receptors. Therefore, ex vivo preincubation of donor marrow cells with IL-3 and GM-CSF may be a useful tactic in bone marrow transplantation.

Animals↗

PubMed1991

Interstitial collagen synthesis by somatic testicular cells in culture.

The synthesis, distribution and types of collagen produced by somatic testicular cells in culture was studied. To investigate whether changes in collagen synthesis correlate with the age of the animal, cultures derived from immature and pubertal rats were established. Immature rats synthesize 40 per cent more collagen than pubertal rats. Both groups of animals synthesize procollagen types I and III. Pro-collagen type I is present in the culture medium as well as in the cell fraction, while type III is only detected in the culture medium. In the transition from immature to pubertal rat, the ratio of procollagen type III to procollagen type I diminishes from 5.7 to 1.7. These results indicate that the synthesis, distribution and molecular characteristics of interstitial collagens changes with the age of the animal. Since, the content of other extracellular matrix components such as proteoglycans and collagen type IV also varies with age, we postulate that the composition of the extracellular matrix in the testes is not constant but changes with sexual development.

Animals↗

PubMed1991

Homing of hemopoietic progenitor cells to the marrow.

The recognition of hemopoietic stem cell after intravenous transplantation of marrow cells occurs initially by a lectin moiety on the surface of marrow sinus endothelium. The cell is then transported across the endothelial cytoplasm much in the way that a soluble ligand, such as transferrin, is transported. In the extravascular compartment, the cell binds to lineage-specific stromal cells. This mechanism, known as homing, is mediated by a lectin-glycoconjugate interaction, the lectin being on the surface of progenitor cell with specificity for galactosyl and mannosyl residues. The binding is subsequently stabilized by membrane-bound proteoglycans, integrin-like receptors, and fibronectin.

Animals↗

PubMed1990

Proteoglycan synthesis by cultured liver endothelium: the role of membrane-associated heparan sulfate in transferrin binding.

Liver endothelium has been reported to possess membrane receptors for the iron-binding protein transferrin (Tf). Similarly, the core protein of proteoglycans (PG) associated with cell membrane in many cell systems can bind Tf. To find out if membrane-associated proteoglycans can explain Tf-binding ability of liver endothelium, we investigated the synthesis and distribution of proteoglycans by isolated, cultured liver capillary endothelium. Cells were isolated and cultured for 48 h in sulfate-free medium and pulse-labeled with 35SO4. The relative distribution of 35SO4-labeled macromolecules, determined in the extracellular (EC), membrane-associated (MA), and intracellular (IC) pools, was respectively 74, 15, and 10%. Membrane-associated proteoglycan (MA-PG) was further purified by ion exchange and gel chromatography. Glycosaminoglycan (GAG) chain characterization indicated about 78% chondroitin sulfate, 7% dermatan sulfate, and about 14% heparan sulfate (HS). Similar GAG chain characterization was made for PG in the EC and IC pools. Transferrin-binding ability of MA-PG was studied by affinity column chromatography, using CNBr-activated sepharose bound to transferrin. About 15% of the labeled MA-PG was specifically bound to Tf-affinity column and could be eluted by excess soluble Tf. This proportion was similar to the proportion of HS in the total membrane-associated pool. Moreover, the eluted labeled material was susceptible to pretreatment with heparitinase, confirming its HS nature. We conclude that the transport capillary endothelium of the liver can synthesize HS proteoglycans which are membrane-associated and this MA-HS pool can bind transferrin. The finding may provide a molecular basis for transferrin binding to liver endothelium and may explain the subsequent transendothelial transport of iron-transferrin complexes into the liver.

Animals↗

PubMed1990

Collagenase-like activity associated to the leukemic WEHI-3B cell line.

The leukemic cell line, WEHI-3B presents a latent collagenase-like activity which is activated after trypsin treatment. The enzymatic activity is not released from cells and is specific for the degradation of interstitial collagens. After attachment of WEHI cells to a hemopoietic stroma derived from long-term bone marrow cultures, the organization of the stroma is disrupted. This effect was not observed after the attachment to stroma of normal progenitor cells. These results suggest that the collagenase-like activity in leukemic cells may contribute through collagen degradation, to the disorganization of the marrow stroma. The latter was confirmed by the use of a labeled-collagen containing stroma, which was degraded when cocultured with WEHI cells, but not with normal progenitor cells.

Animals↗

PubMed1989

Proteoglycan synthesis by hematopoietic progenitor cells.

The synthesis of proteoglycans (PG) by hematopoietic stromal cells has been reported. But PG synthesis by hematopoietic progenitor cells has not been explored. We have studied synthesis, cellular distribution, and molecular characteristics of PG by a cloned interleukin-3 (IL-3)-dependent hematopoietic progenitor cell line, FDCP-1, which is cloned from murine long-term marrow cultures. Under appropriate conditions the cell can differentiate into granulocytes and macrophages, and therefore, can be considered CFU-GM equivalent. The pattern of PG synthesis was studied by 35SO4 labeling. FDCP-1 cells actively synthesize PG, which are distributed in the intracellular, membrane-associated (MP), and extracellular pools. After purification of the 35S-labeled material by ion-exchange and gel filtration techniques, a single chondroitin sulfate-PG (CIS-PG) was observed to be present in the three studied pools. By Sepharose CL-4B chromatography, this PG has a Kav of 0.47, which after alkaline treatment is shifted to a Kav of 0.67. This indicates the proteoglycan nature of the 35SO4-labeled material. The MP CIS-PG is not stable. It is released to the culture medium where it is subsequently processed. However, in the presence of hematopoietic stromal cells D2X, the stability of MP proteoglycan of FDCP-1 cells is enhanced, suggesting that the synthesis of PG by progenitor cells and its accumulation in the membrane may have a role in the interaction between progenitor and stromal cells.

Animals↗

PubMed1989

Comparison of desialylation of rat transferrin by cellular and non-cellular methods.

We have previously shown that the liver endothelium can desialylate the glycoprotein transferrin (Tf). In the present work we provide evidence that asialotransferrin obtained by this means behaves differently on Ricinus communis agglutinin (RCA120) lectin affinity chromatography from asialotransferrin obtained by either neuraminidase treatment or acid hydrolysis. Purified rat transferrin was radiolabelled either with 125I (protein moiety) or with 3H (sialyl residues), and subsequently saturated with iron. It was then passed through an RCA120-agarose column to isolate the fully sialylated component. Sialylated Tf was then desialylated either by incubation with purified rat liver endothelium or, in vitro, by neuraminidase treatment or by acid hydrolysis. The protein was again subjected to RCA120 column chromatography. Although both neuraminidase treatment and acid hydrolysis almost completely desialylated the glycoprotein (as evidenced by near absence of 3H label), the glycoprotein was not retained by the RCA120-agarose column. By contrast, liver endothelium partially desialylated the glycoprotein, but this desialylated fraction was retained by the RCA120-agarose column. These results suggest that desialylation with neuraminidase or acid hydrolysis may be inadequate for functional studies of asialotransferrin.

Animals↗

PubMed1989

Synthesis of proteoglycans and hyaluronic acid by long-term cultures of testicular cells from immature and pubertal rats.

Long-term cultures of somatic testicular cells derived from immature and pubertal rats were used to study the synthesis of proteoglycans (PG) and hyaluronic acid (HA). Labelled PG and HA in the culture medium, membrane-associated and intracellular pools were characterized by gel filtration, ion exchange chromatography and selected enzymatic and chemical treatments. Somatic cells synthesize a PG containing both heparan and chondroitin/dermatan sulfate (CS/DS) chains and a PG containing only CS/DS chains. No major qualitative changes in the type of PG were observed in cells derived from immature and pubertal animals. However, significant age-dependent differences in the cell distribution pattern of PG and HA were determined. This may have implications in the regulation of spermatogenesis.

Animals↗

PubMed1989

Synthesis of extracellular matrix components by somatic testicular cells from immature and pubertal rats.

Total testicular cells derived from immature and pubertal rats were cultured under long-term conditions. Somatic adherent cells proliferated in culture and produced collagen and proteoglycans. Collagen synthesis accounted for 25% and 5% of total protein synthesized by adherent cells derived from immature, and pubertal rats, respectively. Proteoglycan synthesis was higher in cells from immature than from pubertal rats. The proportion of different types of glycosaminoglycan chains (particularly hyaluronic acid and chondroitin sulphate) also varied according to the age of the donor. The results suggest that the synthesis of extracellular matrix components by somatic testicular cells is an age-related process which probably plays an active role in spermatogenesis.

Aging↗

PubMed1989

Changes in collagen synthesis by human bone marrow fibroblasts with progressive subcultivation.

Bone marrow fibroblasts from normal and leukemic patients were used to investigate the relationship between serial subcultivation and changes in collagen synthesis. A regime was established to generate subcultures up to 35 cumulative population doublings (CPDs) in normal cells and to 9 CPDs in leukemic cells. In both types of cells, collagen synthesis decreased as subcultivation progressed. In normal cells, collagen synthesis was reduced to 10% of the original levels at 18 CPDs and in leukemic cells at 8 CPDs. In normal fibroblasts, collagen synthesis was more profoundly affected than overall protein synthesis by subcultivation. In acute lymphoblastic leukemia-derived fibroblasts, the decrease in collagen synthesis paralleled that of total protein.

Bone Marrow↗

PubMed1989

Relationship of the glycan structure of glycoproteins to the desialylation process by rat liver endothelium.

To investigate the variations in desialylation of glycoproteins by liver endothelium, we compared endothelial desialylation for 3 glycoproteins, human ceruloplasmin, human and rat transferrin. Radiolabeled glycoproteins were chased through purified rat liver endothelium and then fractionated by lectin affinity chromatography. Endothelium processed glycoproteins were fractionated by RCA120 chromatography into sialylated and desialylated components. The latter was then studied by Con A chromatography. Desialylation occurred only when the molecule contained at least a single triantennary chain of glycan. Desialylation was minimal in the case of human transferrin which contains mostly biantennary branching pattern. Thus, it appears that a single triantennary glycan chain is necessary and sufficient to trigger desialylation of glycoproteins by liver endothelium and this process is an all-or-none phenomenon.

Animals↗

PubMed1988

[Hematopoietic microenvironment: cellular and extracellular matrix elements].

In bone marrow, cellular stroma together with extracellular matrix (EM) provide an adequate microenvironment for the proliferation and differentiation of hemopoietic progenitor cells. In this article we describe studies on the cell characteristics of a main stromal phenotype, a fibroblast-like cell and its ability to produce in vitro EM components. Comparative studies were performed in fibroblast cultures derived from normal and acute lymphoblastic leukemic (ALL) bone marrow. The grow characteristics of fibroblasts from ALL marrow as well as its capacity to synthetize collagen, fibronectin and GAGs are impaired when compared to fibroblast from normal marrow. Thus, in ALL the impaired production of EM biomolecules by a transient damaged population of stromal cells, may contribute to the development of a defective microenvironment for hemopoiesis.

Bone Marrow Cells↗

PubMed1988

Analysis of the microheterogeneity of the glycan chain of rat transferrin.

To investigate the microheterogeneity of the glycan chain of rat transferrin, either the protein moiety was labeled with 125I or the sialyl residues with 3H. The molecule was then subjected to Con A chromatography. Three components were obtained. Each was enzymatically desialylated and sialyl/protein molar ratios were calculated. The native protein as well as the 3 components were also subjected to isoelectric focusing. The results indicated that rat transferrin may have 3 types of glycan chain: The major type (60%) corresponds to a molecular species with triantennary branching, while 30% consists of molecules with biantennary and 10% with tetraantennary branching. The last species has not been previously described.

Animals↗