Maackia amurensis agglutinin discriminates between normal and chronic leukemic human lymphocytes.

Altered glycosylations of cell surface glycoproteins often accompany malignant transformation and lectins are useful for probing these alterations. Lymphocytes exhibit characteristic surface glycoproteins which serve as markers of cell status and development. The present work was undertaken to compare, on blots, the binding characteristics of membranes isolated from normal peripheral blood lymphocytes and chronic lymphatic leukemia cells to five different lectins, from Datura stramonium, Maackia amurensis, Sambucus nigra, Galanthus nivalis and Peanut. The Maackia amurensis lectin interacted with the normal lymphocytes but showed no binding to malignant cells. Hence, we suggest the Maackia lectin may be used to differentiate normal from leukemic cells.

Interaction of hypothalamic Na,K-ATPase inhibitor with isolated human peripheral blood mononuclear cells.

A ligand for the digitalis receptor located on the membrane-embedded Na,K-ATPase (NKA; EC 3.6.1.37) has been isolated from bovine hypothalamus (hypothalamic inhibitory factor; HIF) and identified as isomeric ouabain (Tymiak et al., 1993, Proc. Natl. Acad. Sci. 90: 8189-8193). In analogy to cardioactive steroids (CS) derived from plants or from toad, HIF inhibits the Na/K-exchange process and the ATPase activity of isolated Na,K-ATPase although by a different molecular action mechanism. In the present work we show that, as plant-derived ouabain, HIF inhibits 86Rb-uptake by isolated human lymphocytes with an IC50 of about 20 nM; above this concentration HIF reduces cell viability in contrast to ouabain. The decrease in cell viability by excess HIF is accompanied by discrete morphological alterations (mitochondrial swelling) visible by transmission electron microscopy of ultra-thin sectioned peripheral blood mononuclear cells. Taken together the results show that the hypothalamic NKA inhibitor blocks NKA of isolated human lymphocytes with high potency at nanomolar concentrations without toxicity; concentrations exceeding the ones required to block 86Rb-uptake reduce cell viability, probably due to leak formation across the NKA molecule. Thus, lymphocytes constitute a potential target for HIF action and by their altered NKA status a possible messenger between the nervous and the immune system.

Interaction of radiolabelled Na,K-ATPase-liposomes with human peripheral blood mononuclear cells.

Artificial phospholipid vesicles (liposomes) containing in their membrane about eight Na,K-ATPase (sodium pump) molecules per vesicle were incubated in the presence of [110mAg]silver nitrate to label the membrane protein; silver binds specifically to the Na,K-ATPase protein. When such silver-labelled liposomes were incubated with freshly isolated human peripheral blood mononuclear cells, a large number of liposomes was found in cells as evidenced by their 110mAg content after washing them with powerful silver chelators. Thus, liposomes containing an integral membrane protein can be transferred to human peripheral blood mononuclear cells rapidly and without toxicity.

Role of cell membrane Na,K-ATPase for survival of human lymphocytes in vitro.

Lymphocytes are primordial immune cells with variable life times. Besides genetic programming, extracellular factors interacting with cell surface receptors might alter cell survival. We investigated whether the activity of the membrane-embedded Na,K-ATPase (EC3.6.1.37) or sodium pump (NKA) plays a role for cell survival since this ubiquitous system establishes the vital transmembrane Na and K gradients as well as the resulting high intracellular K/Na ratio required for macromolecule synthesis; furthermore, the system exposes an extracellular inhibitory receptors for cardioactive steroids and palytoxin. Isolated human lymphocytes were incubated in vitro and their viability assessed by exclusion of trypan blue. Various incubation conditions were compared; in RPMI-1640 medium cell viability was preserved for 30 h at 37 degrees C. Externally added ouabain, a hydrophilic cardioactive steroid, blocked the [86Rb]potassium uptake at nanomolar concentrations. Despite pump inhibition ouabain did not alter lymphocyte survival, even at 10 mM for 30 h. By contrast, the hydrophilic toxin palytoxin, the most potent animal poison described so far, killed all cells within 2 h at 10 nM; this toxin is known to act via the sodium pump and to provoke deadly cation-leaks by unmasking a channel component. Intracellular Na increased and K decreased as measured by atomic absorption spectrometry in presence of palytoxin; cell swelling was seen by electron microscopy. Ouabain protected the cells from the toxic effect of palytoxin. The results reveal a pivotal role of NKA integrity for lymphocyte survival.

Na,K-ATPase and carboxyfluorescein distinctly alter vesicle formation in vitro.

The mechanism of vesicle formation as well as the precise reasons for their stability are not known. Thus, it is necessary to simulate the process in vitro for studying its mechanism. If phospholipids are suspended in physiological solution by means of cholate and the detergent is then removed by dialysis, the phospholipids self-assemble to form unilamellar vesicles. We report here that the addition of Na,K-ATPase (an integral membrane protein) to the phospholipids changes the vesicle structure, they become larger and a multilamellar population appears. By contrast, carboxyfluorescein, a compound commonly used for labelling the aqueous vesicle compartment, produces an unexpected effect on vesicle structure by inducing complex, tore-like intravesicular multilayer formations associated with a 5-fold increase in diameter. Thus, the presence of a protein in the membrane phase or of a compound in the water phase can influence and direct vesicle formation in vitro; these model systems might give some clues to possible physicochemical or biological factors governing the formation of natural membrane structures.

Potent and reversible interaction of silver with pure Na,K-ATPase and Na,K-ATPase-liposomes.

The Na,K-ATPase (EC 3.6.1.37) is the receptor for cardioactive steroids, the only specific inhibitors known at the present time for this unique membrane bound transport system. We report here that silver is the most rapid and potent inhibitor of isolated Na,K-ATPase ever described. Inhibition of Na,K-ATPase activity by silver is immediate and strikingly distinct from other inhibitors: addition of 1 mM of cysteine or DMPS reactivates the silver blocked-enzyme immediately. The results reveal that silver interacts with Na,K-ATPase and inhibits differently by an on-off mechanism involving most likely a few critical sulfhydryl groups. Inhibition of Na-K transport by silver has been demonstrated also in an artificial membrane, e.g., in liposomes reconstituted with pure Na,K-ATPase performing active transport. Silver inhibits the active 86Rb transport mediated by the pure Na,K-ATPase molecule. The Na,K-ATPase contained in the liposomes was labeled specifically with 110mAg and appeared to bind two silver ions. Taken together, the results show that the mechanism of silver interaction with Na,K-ATPase might be different from other metals, for instance, mercury. The unique action mechanism of silver suggests a fundamental role of a few critical sulfhydryl groups for Na,K-transport.

Osteoarthrosis and apatite synovitis. Pathological study of a metacarpophalangeal joint.

A case of metacarpophalangeal osteoarthrosis associated with synovial apatite deposits is reported. The size of the crystals indicates that they have been thickened by a recrystallization process; the latter could have been provoked by Ca and Po4 ions released by dissolution of some apatite crystals brought by calcified debris of bone or cartilage coming from the abraded osteoarthrotic surfaces. The role of such thickened crystals in synovial inflammation is discussed as well as their possible diagnostic value in determining origin and pathogenesis of a given synovial apatite deposit.

Rapidly progressive osteoarthrosis of ochronotic origin. A pathologic study.

A case of hip osteoarthrosis associated with ochronosis in a 65-year-old woman is reported. Characteristic features of both conditions were observed macroscopically and on light and electron microscopic examination. In the cartilage the pigment deposits were located on and between thick collagen fibrils. In the synovial membrane there were embedded packets of cartilage shards of which the collagen fibrils and pigment were phagocytosed, as well as calcified bone debris whose disaggregation might have explained the presence of some apatite deposits free of any underlying collagen structure. As also previously observed, the present case of ochronotic hip osteoarthrosis is remarkable for the minor osteophyte formation and for the inclusion of pigmented cartilage shards in the osteomedullar remodeled territory. It also demonstrates a collapse of the femoral head cortex presumably related to the rapid clinical and radiologic evolution. By the well-known origin of its chondropathy and by the pigment labeling of the cartilage, ochronotic arthropathy provides an almost experimental model for analyzing a broader problem, i.e., that of the various components of an osteoarthrotic remodeling.

Histological study of a case of recurrent olecranon bursitis with mixed calcium pyrophosphate dihydrate and apatite crystal deposits.

This is the report of a light and transmission electron microscopic study of an olecranon bursitis and of the adjacent distal tricipital tendon in an 83 year-old man. The data are compared with those of a similar study in the same patient performed 2 years ago. Calcium pyrophosphate dihydrate crystals were observed in the bursal fluid, in the inner part of the bursal wall (extracellular localization and intracellular phagocytosis) as well as in the peripheral part of the tendon. In addition, small apatite deposits were observed in the bursa and tendon by electron microscopy. The origin of these bursal deposits is discussed; it is suggested that they may be related to an exchange from the tendon to the remodelled bursal wall.