Mehdi Tavassoli

Laboratory Markers of Ethanol Intake and Abuse: A Critical Appraisal

Laboratory markers for ethanol intake and abuse and chronic alcoholism currently in use have been critically reviewed. The merits and pitfalls of each test have been evaluated. The clinical use of the new test of carbohydrate-deficient transferrin has been particularly emphasized. Carbohydrate-deficient transferrin currently provides the highest specificity and sensitivity of all commonly used markers of alcoholism.

Homing of hemopoietic progenitor cells to the marrow.

Abstract 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.

Transendothelial transport (transcytosis) of iron—transferrin complex in the bone marrow

To determine the transport pathway of iron-transferrin complex (Fe-TF) across the marrow-blood barrier, we labeled Fe-TF with colloidal gold and perfused rat femoral marrow with this probe. At 4 degrees C, the probe bound to the luminal surface of marrow sinus endothelium. The binding was inhibitable in the presence of excess native Fe-TF indicating the specificity of the binding. At 37 degrees C, the probe was internalized largely via a system of coated pits and vesicles and transported across the endothelium via a system of tubules and endosomal vesicles. It could not be ascertained if all Fe-TF was still associated with the colloidal gold probe within the endothelium, but the probe appeared to be externalized on the abluminal side into the interstitium where it subsequently bound to the surface of marrow erythroblasts and was internalized. Endothelium appeared to store part of the probe within a large vesicular system. No transport of Fe-TF was noted through diaphragmed fenestrations, diaphragmed vesicles, or interendothelial junctions. No endothelial uptake of this magnitude was noted when native gold particles or gold-labeled bovine serum albumin was used. Our findings indicate that in the bone marrow, gold-labeled Fe-TF is first taken up by sinus endothelium through a receptor-mediated mechanism and is possibly transported transendothelially via a vesicular system (transcytosis).

Review: Transferrin-Mediated Cellular Iron Uptake

The basic model for cellular uptake of iron relies on the iron-chelating protein transferrin (Tf), which is capable of binding iron under one set of conditions and releasing it under another set of conditions. Tf has specific membrane receptors on the surface of the cells that require iron. Tf-receptor binding is followed by internalization through a system of coated pits and vesicles. The rapid decline of pH of these vesicles leads to release and sequestration of iron by the cell. Apotransferrin-receptor complex returns to the cell surface, where, under neutral pH conditions, apotransferrin is dissociated. Other models for cellular uptake of iron include extraction of iron from Tf on the cell surface without internalization, uptake by adsorptive mechanism, and fluid-phase endocytosis. Recent advances in cellular and molecular biology, gene cloning, and monoclonal antibody technique have elucidated many features of these processes at a molecular level. These advances are reviewed and prospects for future work discussed.

Mapping of the bone marrow sinus endothelium with lectins and glycosylated ferritins: Identification of differentiated microdomains and their functional significance

The distribution of lectin binding sites and sugar-recognizing systems (lectin-like substances) was studied on the luminal side of the bone marrow sinus endothelium in rats. Ferritin-conjugated lectins (Con A, PHA, RCA, WGA, UEA) and glycosylated ferritins (mannosyl, fucosyl, chitobiosyl) were used as probes. With the exception of UEA, all lectins bound to the endothelial surface. The binding was heavier on the plasmalemma proper of the nuclear region (PP-N) compared to that of the tapered region (PP-T). Lectin mapping also identified differentiated microdomains with less or no bindings. These domains related to the transport organelles in the endothelium: luminal vesicles (LV) and diaphragmmed fenestrae (DF). The relative density of binding for these four domains demonstrated the following spectrum PP-N greater than PP-T greater than LV greater than DF. The relation of this binding pattern to the transport function of marrow sinus endothelium for various cells and molecules has been discussed. Lectin mapping also identified heavily labeled microvilli and microprojections from the membrane. These organelles may have a function in recognizing cells and molecules for the transport. The absence of binding for UEA, also absent in liver endothelium, may differentiate sinus endothelia from other endothelia. This lectin is thought to be endothelium-specific in other systems. No binding was observed with glycosylated ferritins suggesting the absence of lectin-like substances on marrow sinus endothelium.

In vivo development of adipose tissue following implantation of lipid-depleted cultured adipocyte.

Abstract The monolayer culture of isolated and disaggregated adipocytes from rat omental and perirenal sites, gave rise to a population of fibroblast-like cells, usually devoid of lipid inclusion. Similar fibroblast-like cells have been obtained in cultures of adipose tissue stromal cells and are thought to be undifferentiated adipocyte stem cells. Although the adipocyte-derived fibroblasts were morphologically indistinguishable from culture-derived fibroblasts of other origins, upon autoimplantation into the splenic bed they regained the lipid inclusion and developed again into adipose tissue. The findings suggest that the transformation of adipose cells into fibroblast-like cells is reversible modulation and not a dedifferentiation into the adipose tissue stem cell. This work also substantiates the increasingly recognized heterogeneity of fibroblasts.

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.

Biphasic uptake of iron-transferrin complex by L1210 murine leukemia cells and rat reticulocytes.

The kinetics of the cellular uptake of iron-transferrin complex was studied in L1210 murine leukemia cells and rat reticulocytes using 125I-transferrin. Saturation of transferrin with iron was necessary for optimal uptake. Following the incubation of cells with the radiolabeled complex a biphasic pattern of uptake was observed. The initial phase was rapid and relatively temperature-independent and was not altered by ethylamine, an inhibitor of transglutaminase activity which is necessary for receptor-mediated endocytosis. This phase was considered to result from receptor-ligand interaction which could be reversed to a great degree by replacement with unlabeled transferrin. A plateau was then reached, indicating a saturation of receptors. After 30 min a second phase of uptake was indicated by the second rise in the curve. This phase was slow, relatively temperature-dependent and could be abolished by ethylamine. It was interpreted as evidence of internalization of the ligand. Analysis of the data from competition studies with unlabeled transferrin indicated that the first phase might itself comprise a reversible and an irreversible step with a ratio of 5 to 1.4 for bound transferrin. Thus, the cellular uptake of iron-transferrin complex may consist of a reversible ligand-receptor interaction. Conformational changes may render this interaction irreversible and the internalization of the ligand may then follow.

A minibead method to study cell surface receptors for various proteins coupled to latex particles.

A method is described in which various proteins are linked covalently to amide-modified polystyrene latex minibeads (0.345 μ m). The minibeads are then used as markers to identify, quantitate, and delineate the topography of the protein receptors on the cell surface in scanning electron microscopy (SEM) and to make time sequence observations on their internalization in transmission electron microscopy (TEM). The beads are first activated by glutaraldehyde and then coupled to the protein. Optimal conditions were determined by coupling human serum albumin to the minibeads. Bead activation was best at 37°C when incubated for 24–48 hr with 5% glutaraldehyde. Coupling of the activated beads to the test protein occurred optimally at 22°C and at an incubation time of 6 hr. The recommended protein concentration is 200 μ g/mg minibeads. Using this method, transcobalamin II, transferrin, or insulin linked to minibeads was, respectively, incubated with mouse L1210 cells, rat reticulocytes, and adipocytes. Covalently bound proteins retained their biological activities and from TEM and SEM findings, evidence for receptor-mediated transport process was also obtained. This method provides advantages over the previously used protein markers for localization and delineation of the topography of cell surface receptors, in that (a) it permits covalent binding between protein and the marker, (b) the derivatized protein is structurally stable and retains its biologic activity, (e) the beads are submicrometer in size and permit the delineation of topography of receptors in SEM, and (d) the small size of the beads also permits observations on their internalization in TEM.

The role of liver endothelium in the binding and uptake of ceruloplasmin: Studies with colloidal gold probe

To determine the mode of uptake of ceruloplasmin (CP) by liver, the protein was labeled with colloidal gold and infused into the portal vein. In cold almost all probes bound to the sinusoidal endothelium, and at 37 degrees C internalization via a system of coated pits and vesicles occurred. Only rarely did the probe appear to bypass the endothelium, moving to the abluminal side through the gaps between endothelial cells. In the endothelial cytoplasm, the probe was seen in coated vesicles, endosomes, tubules, and large vesicles which may have formed by fusion of endosomes and tubules. Moreover, externalization of the probe to the abluminal side was noted, and this also occurred via a system of coated vesicles. The findings suggest that the uptake of CP in the liver may be primarily a transendothelial phenomenon (transcytosis).