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Biomedical subjects

B M Anner

Publications and source records attributed to B M Anner.

At least 19 recordsLinked to original sources
PubMed1999

Uptake of reconstituted Na,K-ATPase vesicles by isolated lymphocytes measured by FACS, confocal microscopy and spectrofluorometry.

Na,K-ATPase (EC 3.6.1.37, Na,K-ATPase) is a fundamental vital membrane transport and receptor system which, after biosynthesis, is exported to the plasma membrane in inside-out vesicles. Na,K-ATPase can be extracted form the natural membrane and inserted into artificially formed phosphatidylcholine vesicles (liposomes). The ultrastructure of the reconstituted vesicles has been fully described. In the present work, the Na,K-ATPase-vesicles were labeled with fluorescent tracers either in their water or membrane phase, incubated with freshly isolated human lymphocytes, and the resulting cellular fluorescence measured with fluorescence activated cell sorting (FACS), confocal microscopy and spectrofluorometry. The FACS data show that all lymphocytes take up Na,K-ATPase-vesicles in a dose- and temperature-dependent fashion. Three-dimensional analysis of the fluorescence by confocal microscopy reveals that the fluorescence is contained within the cells. Quantitative determination by spectrofluorometry indicates that depending on the vesicle/cell ratio, a single lymphocyte takes up 650 to 36,500 vesicles within 30 min at 37 degrees C together with up to about 200,000 renal Na,K-ATPase molecules.

Cell Membrane↗
PubMed1999

Inhibition of HIV-1 infection by zinc group metal compounds.

Thirty-seven metal compounds were examined for inhibitory activities against infection with human immunodeficiency virus type 1 (HIV-1). Zinc group metal compounds, namely, zinc acetate, zinc chloride, zinc nitrate, cadmium acetate and mercury chloride, showed anti-HIV-1 activities. Cadmium and mercury compounds at 1-10 microg/ml and zinc compounds at 100 microg/ml strongly inhibited HIV-1 infection, although the cadmium, mercury and zinc compounds had severe cytotoxities at 100, 100 and 1000 microg/ml, respectively. They inhibited transcription of HIV-1 RNA and HIV-1 production at concentrations at which they did not affect the growth of HIV-1-producing cells. They had little effect on syncytium formation resulting from cocultivation of uninfected with HIV-1-producing cells. Nor did they affect HIV-1 DNA synthesis following HIV-1 infection. The metal compounds may owe their anti-HIV-1 effects to inhibition of HIV-1 DNA to RNA transcription, rather than inhibition of the adsorption, penetration or reverse transcription step of HIV-1 infection.

Antiviral Agents↗
PubMed1999

Characterisation of tissue-specific oligosaccharides from rat brain and kidney membrane preparations enriched in Na+,K+-ATPase.

The organ-specific nature of the glycosylation of Na+,K+-ATPase-enriched preparations from kidney and brain tissues has earlier been indicated by the use of lectin-staining techniques. Na+,K+-ATPase is ubiquitous and abundant, and subject to upregulation during cell-division and in certain pathological conditions. Lectins specific for the different carbohydrates displayed by the Na+,K+-ATPases may, therefore, be useful carriers/mediators in tissue-specific targeting. N-linked oligosaccharides purified from Na+,K+-ATPase-enriched preparations from rat brain and kidney were consequently characterised in detail in this study using weak anion exchange and normal phase HPLC (combined with serial glycosidase digestions) and matrix-assisted laser desorption/ionisation mass spectrometry. The oligomannose series of glycans were most abundant in the brain tissue preparation and this contrasted with the renal-associated oligosaccharides that were dominated by families of tetra-antennary glycans (with/without a core fucose) with up to four lactosaminylglycan residues in either branched or linear formation.

Animals↗
PubMed1997

Transposing results from an artificial minicell to a real cell. Experimental evidence for a working hypothesis linking Na,K-ATPase permeability states to specific alterations of cell life.

The present work sets in parallel data obtained with the Na,K-ATPase in artificial vesicular membranes (artificial minicells) and in peripheral human lymphocytes ex vivo. The Na,K-ATPase was purified and reconstituted into single-walled, tight liposomes filled with a reservoir of ATP and Na in which the Na,K-ATPase functions as in cells, that is, the receptor is accessible on the liposome surface (artificial minicells). In this system, an E2-ouabain state impermeable to Rb ions and an Na,K-ATPase-palytoxin state leaky for Rb ions were characterized. The tight E2-ouabain form preserves the viability of isolated lymphocytes, whereas the leaky Na,K-ATPase-palytoxin induces rapid cell bursting and death by a ouabain-sensitive mechanism. Thus, the effect of Na,K-ATPase inhibitors on lymphocyte survival can be predicted from permeability measurements in artificial minicells as verified also with the leak-inducing metals mercury and silver.

Acrylamides↗
PubMed1997

Endogenous natriuretic factors 7: biospecificity of a natriuretic gamma-tocopherol metabolite LLU-alpha.

The structural elucidation and mechanism of action of a potential component, LLU-alpha, of what is possibly a multifactorial complex known as "natriuretic hormone" was recently reported [Wechter, W.J. et al. (1996a) Proc. Natl. Acad. Sci. U.S.A. 93: 6002-6007]. "Natriuretic hormone," a long-sought factor, is believed to regulate extracellular fluid volume and consequently be pathomimetic for hypertension, cirrhosis, congestive heart failure and other volume expanded states. The studies reported herein further characterize LLU-alpha. The precursor of the endogenous LLU-alpha was demonstrated to be gamma-tocopherol by radiolabeling studies. The pharmacokinetics of infused rac-LLU-alpha proved to be biphasic (half-lives: 12 min and 6 h). Specificity of the inhibition of the 70 pS potassium channel of the thick ascending limb of the loop of Henle was examined with the natural S-enantiomer being the most potent known inhibitor whereas the analogous alpha-tocopherol metabolite, rac-5-Me-LLU-alpha, showed no inhibition. Rac-LLU-alpha does not inhibit two isozymes of the Na+/K+-ATPase. LLU-alpha is natriuretic acting via inhibition of the 70 pS potassium channel and not Na+/K+-ATPase, the assumed mechanism of action of the "natriuretic hormone." LLU-alpha, a metabolite of a vitamin, if it were found to play a role in the regulation of extracellular fluid volume, would be the second example of a vitamin acting as a precursor for a hormone. Of considerable interest is the fact that this manuscript reports the first biological activity of gamma-tocopherol, a member of the vitamin E complex.

Animals↗
PubMed1995

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.

Cell Membrane↗
PubMed1995

Normal sensitivity of Na+/K(+)-ATPase isolated from brain and kidney of spontaneously hypertensive rats to sodium, ouabain or mercury.

Genetically hypertensive rats are excellent animal models for investigating putative Na+/K(+)-ATPase alterations associated with the disease. Highly purified Na+/K(+)-ATPase preparations from these animals have not yet been examined. Na+/K(+)-ATPases of two strains of spontaneously hypertensive rats, the Milan hypertensive strain (MHS) and the spontaneously hypertensive rat (SHR) were characterized in comparison with enzymes isolated from their matched normotensive controls; the sensitivity to Na ions as well as the shape and span of the inhibition curves for ouabain and mercury of the isolated Na+/K(+)-ATPases were compared. No functional changes between the purified 'normotensive' and 'hypertensive' Na+/K(+)-ATPases from brain and kidney were detected ruling out drastic structural alterations of the transport system in these two organs of diseased animals.

Animals↗
PubMed1995

Major organ-specific glycoproteins in isolated brain and kidney membranes identified as Na,K-ATPase subunits by combined glycan-, lectin-, and immunoblotting.

In the present work combined glycan-, lectin-, and immunoblotting of isolated brain and kidney membranes shows that the alpha and beta subunits of Na,K-ATPase are the most abundant glycoproteins. Further, Datura stramonium and Galanthus nivalis agglutinins recognize the Na,K-ATPase subunits in a mutually exclusive manner in membranes from human, rabbit and rat brain or human, rabbit, rat, pig and dog kidney indicating the presence of species-independent organ-typical glycoforms. The glycosylation status is not related to the ouabain-sensitivity. Taken together, the data reveals organ-specific glycoforms of Na,K-ATPase which might have roles for organ identification and recognition.

Animals↗
PubMed1994

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.

Cell Separation↗
PubMed1994

Identification of organ-specific glycosylation of a membrane protein in two tissues using lectins.

Since glycosylation of proteins is performed by the host cell, and variable sugar groupings can confer heterogeneity on the same polypeptide, we wished to see whether membrane proteins, in particular the ubiquitous transmembrane Na,K-ATPase, could be glycosylated differently in different organs. Using a highly sensitive enzyme-linked antibody detection system of bound digoxigenin-labelled lectins on nitrocellulose sheets containing electroblotted alpha and beta subunits of kidney and brain Na,K-ATPase, isolated from various rat strains, in combination with isoform-specific immunoblots, we discovered that brain Na,K-ATPase was highly mannosylated in contrast to renal Na,K-ATPase. Thus, we describe the existence of organ-related glycoforms of an integral ubiquitous membrane protein, i.e. diversification of the same polypeptide by organ-typical sugars. At the same time, the presence of the same glycosylation pattern can make distinct protein isoforms occurring in a same organ more homogeneous. Such organ-related glycoforms may serve for tissue identification and as tissue-specific receptors.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

Internalization of poly(D,L-lactic acid) nanoparticles by isolated human leukocytes and analysis of plasma proteins adsorbed onto the particles.

The objective of this work was to investigate the interactions of poly(D,L-lactic acid) nanoparticles prepared by a recently developed salting-out process, with lymphocytes and monocytes isolated from healthy human donors. Nanoparticles were labeled with a hydrophobic fluorescent dye and incubated with lymphocytes and monocytes, and their uptake was followed by flow cytometry in the presence and absence of plasma. Plasma protein adsorption increased nanoparticle uptake by monocytes, whereas a decrease of cellular binding of the nanoparticles to lymphocytes was noted. The cellular uptake for both cell types consisted in a passive adsorption and in an energy-requiring process, because the cells became 2-3 times more fluorescent when the incubation temperature was increased from 4 to 37 degrees C. When nanoparticles were coated with polyethylene glycol 20,000, uptake by monocytes decreased by 43 and 78% in phosphate-buffered saline and plasma, respectively; a similar decrease in nanoparticle uptake was observed for lymphocytes. Two-dimensional gel electrophoresis was performed to identify the plasma opsonins adsorbed onto the nanoparticle surface. Protein mappings for uncoated and polyethylene glycol-coated nanoparticles differed for two spot series. These spots, not yet clearly identified, may represent specific apolipoproteins involved in the metabolism of human lipoproteins, indicating the possible involvement of specific receptors in the uptake of the nanoparticles.

Adsorption↗
PubMed1994

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.

Acrylamides↗
PubMed1994

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.

Cell Survival↗
PubMed1993

Use of two-sided bifunctional liposomes in the study of a hypothalamic Na,K-ATPase inhibitor.

Two-sided bifunctional (ATP-filled) Na,K-ATPase liposomes have been developed as a result of knowledge about the average liposome diameter and volume, the liposome size distribution, the average number of Na,K-ATPase molecules reconstituted per liposome, and the orientation of the reconstituted Na,K-ATPase molecules. The addition of 5-10 microM external 86Rb to the liposomes containing 50 mM encapsulated ATP provoked an impressive 86Rb accumulation by the cell-like-oriented pumps. The successive addition of external ATP activated the pumps in the reversed orientation of the same liposome, leading to total extrusion of the previously accumulated 86Rb. An inhibitor extracted from bovine hypothalamus (hypothalamic inhibitory factor) inhibited the cell-like-oriented population, i.e., acted like an extracellular inhibitor at 30 nM. Conversely, at 75 nM, the reversed pump population was also blocked, indicating that the inhibitor either transversed the membrane or was able to act also at the intracellular enzyme side at a higher concentration. Thus, the side of action as well as the membrane permeability of structurally unknown endogenous Na,K-ATPase inhibitors can be determined simultaneously in a single suspension of two-sided bifunctional Na,K-ATPase liposomes.

Animals↗
PubMed1992

Cysteine protects Na,K-ATPase and isolated human lymphocytes from silver toxicity.

Metal-binding proteins are important components of retroviruses such as human immunodeficiency virus (HIV). Therefore, metals could be used as antiviral agents. However, most metals are toxic for humans with the exception of silver which is toxic only to prokaryotic cells and viruses. In addition, HIV infection causes a decrease in body cysteine. We formed a complex of silver and cysteine, named silver-cysteine. Healthy human lymphocytes were incubated with silver-nitrate or silver-cysteine. Negligible cell survival was seen at 50 microM silver-nitrate. However, in presence of 1 mM cysteine, the viability remained unaffected up to 1 mM of silver. Further, silver inhibition of isolated Na,K-ATPase was easily reversed by cysteine. Thus, non-toxic silver-cysteine could be used as an anti-viral and cysteine-replenishing agent.

Animals↗