bluuebunny/crossref_metadata_2025 huggingface dataset:
Cation‐dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one‐step gradient centrifugation
Authors: Lernmark Årke , Nielsen David A. , Steiner Donald F.
Published: May 2005

A plasma membrane‐enriched fraction was prepared from homogenized rat pancreatic islets by a one‐step sucrose gradient centrifugation. Using 125I‐wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5′‐nucleotidase, Mg2+‐ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3‐0‐methyl‐fluoresceinphosphate was stimulated by K+ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP‐γ‐32P in the presence of MgCl2 was of high specific activity and was optimum at pH 7.0 and 8.2. K+ did not affect ATP‐hydrolysis. At pH 8.2, a small fraction of the total Mg2+‐ATPase activity was inhibited by ouabain in the presence of Na+ and K+. Since K+‐stimulated phosphatase activity does not correlate with Mg2+‐ATPase, the two assay systems define separate enzymatic processes.
Membrane alterations in irreversibly sickled cells: Hemoglobin–membrane interaction
Authors: Lessin Lawrence S. , Kurantsin‐Mills Joseph , Wallas Charles , Weems Henri
Published: May 2005

Irreversibly sickled cells (ISCs) are sickle erythrocytes which retain bipolar enlongated shapes despite reoxygenation and owe their biophysical abnormalities to acquired membrane alterations. Freeze‐etched membranes both of ISCs produced in vitro and ISCs isolated in vivo reveal microbodies fixed to the internal (PS) surface which obscure spectrin filaments. Intramembranous particles (IMPs) on the intramembrane (PF) surface aggregate over regions of subsurface microbodies. Electron microscopy of diaminobenzidine‐treated ISC ghosts show the microbodies to contain hemoglobin and/or hemoglobin derivatives. Scanning electron microscopy and freeze‐etching demonstrate that membrane–hemoglobin S interaction in ISCs enhances the membrane loss by microspherulation. Membrane‐bound hemoglobin is five times greater in in vivo ISCs than non‐ISCs, and increases during ISC production, paralleling depletion of adenosine triphosphate. Polyacrylamide gel electrophoresis of ISC membranes shows the presence of high‐molecular‐weight heteropolymers in the pre–band 1 region, a decrease in band 4.1 and an increase in bands 7, 8, and globin. The role of cross‐linked membrane protein polymers in the generation of ISCs is discussed and is synthesized in terms of a unified concept for the determinants of the genesis of ISCs.
Tight junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone
Authors: Porvaznik Martin , Johnson Ross G. , Sheridan Judson D.
Published: May 2005

Freeze‐fracture and thin‐section methods were used to study tight junction formation between confluent H4‐II‐E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P‐fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E‐face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10−6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.
Solubilization and conversion of hepatic adenylate cyclase to a form requiring MnATP as substrate
Authors: Londos Constantine , Lad Pramod M. , Nielsen Thor B. , Rodbell Martin
Published: May 2005

A general feature of membrane‐bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane‐bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane‐bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane‐bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by Pi or PPi, and (4) and apparent loss of the Me2+‐reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane‐bound form.
Interaction between glycophorin and phospholipids in recombined systems
Authors: Romans Alice Y. , Yeagle Philip L. , O'connor Sally E. , Grisham Charles M.
Published: May 2005

Both the MN‐glycoprotein from human erythrocytes and the hydrophobic fragment from the protein isolated with trypsin treatment, T(is), have been recombined with egg phosphatidylcholine in bilayers at various phospholipid/protein ratios. In order to investigate the effect of the protein on the phospholipid headgroups, 31P nuclear magnetic resonance spectra were obtained with the MN‐glycoprotein recombined with egg phosphtidylcholine, which revealed two classes of phospholipid enviroments, one immobilized and one not immobilized. Electron spin resonance (ESR) of fatty acid methyl ester spin labels provided supporting evidence.

Computer analysis of the ESR spectra indicate that 4–5 moles of phospholipid are immobilized per mole of protein over a wide range of lipid‐to‐protein ratios. The immobilization of the phospholipids appears mediated by both the polar headgroups and the hydrocarbon tails of the phospholipid.
Role of bound water in biological membrane structure: Fluorescence and infrared studies
Authors: Schneider Allan S. , Middaugh C. Russell , Oldewurtel Mary D.
Published: May 2005

Bound water is a major component of biological membranes and is required for the structural stability of the lipid bilayer. It has also been postulated that it is involved in water transport, membrane fusion, and mobility of membrane proteins and lipids. We have measured the fluorescence emission of membrane‐bound 1‐anilino‐8‐naphthalenesulfonate (ANS) and the infrared spectra of membranes, both as a function of hydration. ANS fluorescence is sensitive to polarity and fluidity of the membrane‐aqueous interface, while infrared absorption is sensitive to the hydrogen bonding and vibrational motion of water and membrane proteins and lipids. The fluorescence results provide evidence of increasing rigidity and/or decreasing polarity of the membrane‐aqueous interface with removal of water. The membrane infrared spectra show prominent hydration‐dependent changes in a number of bands with possible assignments to cholesterol (vinyl CH bend, OH stretch), protein (amide A, II, V), and bound water (OH stretch). Further characterization of the bound water should allow its incorporation into current models of membrane structure and give insight into the role of membrane hydration in cell surface function.
Ribosome crystallization in nuclei of chick embryos
Authors: Barbieri Marcello
Published: May 2005

Ribosome crystallization within nuclei has been studied in chick embryos with procedures which increase its frequency by various orders of magnitude as compared to previous findings. The extrusion of ribosome microcrystals from nuclei is reported for the first time, and a model for the transfer of ribosomes from nucleus to cytoplasm is proposed.
Comparison of the major polypeptides of the erythrocyte nuclear envelope
Authors: Cochran David L. , Egle Patsy M. , Shelton Keith R. , Yeoman Lynn C.
Published: May 2005

The three most abundant nonhistone polypeptides (molecular weights 75,000, 71,000 and 61,000) of the avian erythrocyte nucleus have previously been isolated in the nuclear envelope fraction. They have been separated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis and peptide‐mapped after limited enzymatic digestion. Three enzymes–chymotrypsin, papain and Staphylococcus aureus protease–were used. Results obtained with each enzyme indicate strong similarities between the three nuclear envelope polypeptides. The amino acid compositions of the two most abundant polypeptides (P75 and P71) have been determined and found to be similar. Further, they readily yield large fragments upon brief alkaline hydrolysis. For both P75 and P71 the degree and the pattern of alkaline fragmentation are almost identical. A 61,000‐dalton polypeptide which appears to be P61 is obtained from P75 and P71 by mild acid hydrolysis. These results establish the close chemical similarity of these predominant polypeptides in the erythrocyte nucleus and suggest that they serve related functions.
Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells
Authors: Krieger Monty , Anderson Richard G. W. , Goldstein Joseph L. , Brown Michael S. , Smith Louis C. , Kao Yin J. , Pownall Henry J. , Gotto Antonio M.
Published: May 2005

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3‐pyrenemethyl‐23, 24‐dinor‐5‐cholen‐22‐oate‐3β‐yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane‐extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor‐mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence‐activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.
Human fibrosarcoma cells produce fibronectin‐releasing peptides
Authors: Keski‐Oja Jorma , Marquardt Hans , De Larco Joseph E. , Todaro George J.
Published: May 2005

A sensitive radioimmunoassay, specific for human fibronectin, was used to measure the ability of certain biologically active polypeptides to release fibronectin from cultured human lung fibroblasts into their culture media. Concentrated, serum‐free culture supernatant from a human fibrosarcoma cell line was fractionated by gel filtration chromatography in the presence of acetic acid. Various polypeptides with molecular weights between 46,000 and 6,000 were tested for their ability to release fibronectin from cells. The column fraction, containing polypeptides with an apparent molecular weight of 10,000, exhibited the ability to rapidly release fibronectin from target cells. The activity could be inhibited by phenylmethyl sulphonylfluoride. Several other hormonal factors, tested in parallel with the column fractions, failed to show this effect. The 10,000 dalton molecular weight polypeptides may represent a family of cellular gene products responsible for maintenance of low levels of surface associated fibronectin in fibrosarcoma cells and thus be related to their infiltrating properties by preventing the formation of the extracellular matrix.
Fibronectin associated with the glial component of embryonic brain cell cultures
Authors: Kavinsky Clifford J. , Garber Beatrice B.
Published: May 2005

In a basic approach to investigations of neuronal–glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti‐GFA serum.

When unfractionated cerebrum cells dissociated from 10‐day chick or 14‐day mouse embryos were plated as monolayers and cultured for 1‐14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane‐associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal–glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti‐fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti‐GFA or anti‐fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal–glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal–glial interactions is discussed.
Stimulation of human vascular endothelial cell growth by a platelet‐derived growth factor and thrombin
Authors: Zetter Bruce R. , Antoniades Harry N.
Published: May 2005

Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet‐derived growth factor (PDGF) and the protease thrombin. When 104 HuE cells are seeded as a dense island (2‐mm diameter) in the center of a 16‐mm tissue culture well in medium containing 20% human serum derived from platelet‐poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 × 104. When human thrombin (1 μg/ml) is added along with the PDGF, the cell number rises to 9.2 × 104. Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet‐derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.
Effect of thiourea and substituted thioureas on dynein ATPase and on the turbidity response of tetrahymena cilia
Authors: Blum J. J. , Hayes A.
Published: May 2005

The effects of thioura and of several substituted thioureas–phenylthiourea, α‐naphtylthiourea, metiamide, and burimamide–on dynein ATPase have been studied. The substituted thioureas are over 30 times more potent than thiourea in causing enhancement of 30S dynein ATPase activity and inhibition of 14S dynein ATPase activity. The effects of thiourea and phenylthiourea can be prevented by very low concentrations of β‐mercaptoethanol or dithiotheritol. Axonemal ATPase is also enhanced by the thioureas, but the reaction proceeds more slowly than for solubilized 30S dynein. Enhancement of 30S dynein ATPase by metiamide is prevented by low (∼ 1 μM) concentrations of ATP and, less effectively, by AMP‐PNP, but not by AMP‐PCP even though the latter is a stronger inhibitor of 30S dynein ATPase than is AMP‐PNP.

The thioureas inhibit the ATP‐induced decrease in turbidity (measured as ΔA350) of axonemal suspensions. Inhibition of the turbidity response is also prevented by low concentrations of β‐mercaptoethanol, but, in contrast to the irreversible enhancement of ATPase activity, inhibition of the turbidity response is largely reversible. The ability of 30S dynein to rebind onto twice extracted axonemes is not changed by treatment with phenylthiourea or metiamide.

These observations indicate that the thioureas react with at least two sets of SH or S–S groups on axonemes. Reaction with the group(s) on the 30S dynein causes an apparently irreversible enhancement of ATPase activity. Reaction with another group(s) causes a reversible inhibition of the turbidity response.
Membrane dynamics of differentiating cultured embryonic chick skeletal muscle cells by fluorescence microscopy techniques
Authors: Elson Hannah Friedman , Yguerabide Juan
Published: May 2005

Changes in membrane fluidity during myogenesis have been studied by fluorescence microscopy of individual cells growing in monolayer cultures of embryonic chick skeletal muscle cells. Membrane fluidity was determined by the techniques of fluorescence photobleaching recovery (FDR), with the use of a lipidsoluble carbocyanine dye, and by fluorescence depolarization (FD), with perylene used as the lipid probe. The fluidity of myoblast plasma membranes, as determined from FPR measurements in membrane areas above nuclei, increased during the period of myoblast fusion and then returned to its initial level. The membrane fluidity of fibroblasts, also found in these primary cultures, remained constant. The fluidity in specific regions along the length of the myoblast membrane was studied by FD, and it was observed that the extended arms of the myoblast have the highest fluidity on the cell and that the tips at the ends of the arms had the lowest fluidity. However, since the perylene probe used in the FD experiments appeared to label cytoplasmic components, changes in fluidity measured with this probe reflect changes in membrane fluidity as well as in cytoplasmic fluidity. The relative change in each of these compartments cannot yet be ascertained. Tips have specialized surface structures, filopodia and lamellipodia, which may be accompanied by a more immobile membrane as well as a more rigid cytoplasm. Rounded cells, which may also have a more convoluted surface structure, show a lower apparent membrane fluidity than extended cells.
Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release
Authors: Smith Thomas C. , Levinson Charles
Published: May 2005

Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium‐labeled 4,4′‐diisothiocyano‐1,2′‐dihenylethane‐2,2′‐disulfonic acid (3H2DIDS) at 4°C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of 3H2DIDS‐label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the 3H2DIDS‐labeled components of the cell surface. Approximately 73% of the cell‐associated radioactivity is spontaneously released from the cells after 5 h at 23°C. The kinetics of release is consistent with the first‐order loss of two fractions; a slow (τ½ = 360 min) component representing 33% of the radioactivity, and a fast (τ½ = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 μ/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinectics of release to that of a single component (τ½ = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously los in 1 h. However, trypsinization following a 2‐h period of spontaneous release yields material of reduced (43%) SA. Neither 3H2DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.
7‐Acetylcytochalasin B: Differential effects on sugar transport and cell motility
Authors: Lees Andrew , Lin Shin
Published: May 2005

Cytochalasin B (CB) is a potent inhibitor of sugar transport and cell motility in animal cells. We have synthesized and characterized the CB derivative 7‐acetylcytochalasin B (CBAc) and have found that it has differential effects on transport and motile processes in fibroblasts. The derivative inhibited sugar transport in human red cells, 3T3 cells, and chicken embryo fibroblasts at micromolar concentrations, although it was less potent than its parent compound. Unlike CB, which causes fibroblasts to round up and arborize at less than 10 μM, CBAc had no effect on fibroblast morphology and membrane ruffling at concentrations as high as 90 μM. Competitive binding experiments using [3H] CB showed that the affinity of CBAc for sites related to sugar transport in the red cell membrane is about one‐fourth of that of CB. In contrast, similar experiments using [3H] dihydrocytochalasin B (a derivative which inhibits cell motility but not sugar transport) showed that the affinity of CBAc for sites associated with red cell spectrin and actin is only about 1/20 of that dihydrocytochalasin B. This study demonstrates that acetylation of the C‐7 hydroxyl group of CB reduces its effect on cell morphology and motility much more than its ability to inhibit sugar transport. This observation, together with our earlier work with dihydrocytochalasin B, establishes that the pharmacologic effects of CB on fibroblasts result from the binding of the drug to two distinct classes of receptors and that these receptors interact with different parts of the cytochalasin molecule.
Action of phorbol esters in cell culture: Mimicry of transformation, altered differentiation, and effects on cell membranes
Authors: Weinstein I. Bernard , Lee Lih‐Syng , Fisher Paul B. , Mufson Alan , Yamasaki Hiroshi
Published: May 2005

The carcinogenic process is usually multifactor in its causation and multistep in its evolution. It is likely that entirely different molecular mechanisms underlie the many steps in this process. In contrast t o initiating carcinogens, the action of the tumor‐promoting phorbol esters does not appear t o involve covalent binding t o cellular DNA and they are not mutagenic. Recent studies in cell culture have revealed two interesting biologic effects of the phorbol esters and related macrocyclic plant diterpenes. The first is that at nanomolar concentrations they induce several changes that resemble those seen in cells transformed by chemical carcinogens or tumor viruses. These include altered morphology and increased saturation density, altered cell surface fucose‐glycopeptides, decrease in the LETS protein, increased transport of deoxyglucose, and increased levels of plasminogen activator and ornithine decarboxylase. In transformed cells exposed to phorbol esters the expression of these features is further accentuated. Phorbol esters do not induce normal cells to grow in agar but they do enhance the growth in agar of certain transformed cells. The second effect of the phorbol esters is inhibition of terminal differentiation. This effect extends to a variety of programs of differentiation and is reversible when the agent is removed. With certain cell culture systems induction of differentiation, rather than inhibition, is observed. Both the transformation mimetic and the differentiation effects are exerted by plant diterpenes that have tumor‐promoting activity but not by congeners that lack such activity. The primary target of phorbol esters appears to be the cell membrane. Early membrane‐related effects include enhanced uptake of 2‐deoxyglucose and other nutrients, altered cell adhesion, induction of arachidonic acid release and prostaglandin synthesis, inhibition of the binding of epidermal growth factor t o cell surface receptors, altered lipid metabolism, and modifications in the activities of other cell surface receptors. A model of “two stage” carcinogenesis encompassing the known molecular and cellular effects of initiating carcinogens and tumor promoters is presented. According to this model, initiating carcinogens induce stable alterations in the cellular genome but these are not manifested until tumor promoters modulate programs of gene expression and induce the clonal outgrowth of the initiated cell.
Introduction of metastatic heterogeneity by short‐term in vivo passage of a cloned transformed cell line
Authors: Talmadge James E. , Starkey Jean R. , Davis William C. , Cohen Arthur L.
Published: May 2005

An experimental system for the study of metastasis has been developed using an epithelioid cell line of hepatic origin which had previously been chemically transformed in vitro. These metastatic cells were studied in the syngeneic rat strain. The cloned parent cell line metastasizes only to the lungs following in travenous, subcutaneous, or intraperitoneal injection. The metastatic phenotype is stable during in vitro passage, and subclones from the parent clone have a metastatic capacity statistically similar to that of the parent clone. Following ascites passage of the parent cell line, the cell population obtained exhibits the same metastatic ability as the parent clone. However, subclones obtained from the ascites‐passaged population exhibit metastatic heterogneity. This heterogeneity is introduced by the host passage and not by in vitro culture or subcloning. In the case of the two metastatic variants examined, the difference in the metastatic phenotype is found not to be due to differences in arrest or trapping of the cells but appears to be related to long‐term survival and proliferation of the tumor cells following their arrest in the lungs. Morphologically the variants are very similar, and growth of the metastatic foci provokes a vigorous inflammatory response by the host.
An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts
Authors: Furcht L. T. , Wendelschafer‐Crabb G. , Mosher D. F. , Foidart J. M.
Published: May 2005

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti‐peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non‐periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.
Light‐driven primary sodium ion transport in halobacterium halobium membranes
Authors: Lanyi Janos K.
Published: May 2005

Light‐induced sodium extrusion from H halobium cell envelope vesicles proceeds largely through an uncoupler‐sensitive pathway involving bacteriorhodopsin and a proton/sodium antiporter. Vesicles from bacteriorhodopsin‐negative strains also extrude sodium ions during illumination, but this transport is not sensitive to uncouplers and has been proposed to involve a light‐energized primary sodium pump. Proton uptake in such vesicles is passive, and under steady‐state illumination the large electrical potential (negative inside) is just balanced by a pH difference (acid inside), so that the protonmotive force is near zero. Action spectra indicated that this effect of illumination is attributable to a pigment absorbing near 585 nm (of 568 for bacteriorhodopsin). Bleaching of the vesicles by prolonged illumination with hydroxylamine results in inactivation of the transport; retinal addition causes partial return of the activity. Retinal addition also causes the appearance of an absorption peak at 588 nm, while the absorption of free retinal decreases. The 588 nm pigment is present in very small quantities (0.13 nmole/mg protein), and behaves differently from bacteriorhodopsin in a number of respects. Vesicles can be prepared from bacteriorhodopsin‐containing H halobium strains in which primary transport for both protons and sodium can be observed. Both pumps appear to cause the outward transport of the cations. The observations indicate the existence of a second retinal protein, in addition to bacteriorhodopsin, in H halobium, which is associated with primary sodium translocation. The initial proton uptake normally observed during illumination of whole H halobium cells may therefore be a passive flux in response to the primary sodium extrusion.
Visualization of the turkey erythrocyte β‐adrenergic receptor
Authors: Durieu‐Trautmann O. , Delavier‐Klutchko C. , Vauquelin G. , Strosberg A. D.
Published: May 2005

We have recently described the affinity chromatography purification of the turkey erythrocyte β‐adrenergic receptor. The minute amounts obtained initially precluded extensive biochemical characterization. To improve the yield of the receptor, the erythrocyte membranes have been prepared by a new method. This procedure resulted in a 10‐fold higher receptor density in comparison with the membrane preparation used previously. The new membranes also contained a catecholamine‐sensitive guanine triphosphatase and an adenylate cyclase sensitive to Gpp(NH)p and l‐epinephrine. Solubilization by a double digitonin extraction resulted in a preparation containing 4–6 pmoles of 3H‐dihydroalprenolol binding sites per mg of membrane protein.

A single step of affinity chromatography on alprenolol‐sepharose of the soluble digitonin extract resulted in an additional 1,000‐fold purification of the receptor. The overall purification factor was 20,000 relative to the binding activity of the crude membrane preparations.

Electrophoresis in SDS‐polacrylamide of iodinated purified β‐receptors revealed, after autoradiography, the presence of four major components. Three of these, corresponding to molecular weights of 170,000, 33,000, and 30,000, respectively, were not affected by reduction with β‐mercaptoethanol and were not observed when the digitonin extracts were loaded on the affinity gel in the presence of an excess of l‐propranolol. A fourth 52,000‐dalton component (60,000 daltons after reduction with β‐mercaptoethanol) remained apparent even when affinity purification was prevented by addition of l‐propranolol.

Our results suggest that the β‐adrenergic receptor is composed of at least three subunits that interact by noncovalent bonds.
Induction and long‐term maintenance of Thy‐1 positive T lymphocytes: Derivation from continuous bone marrow cultures
Authors: Tertian Gérard , Yung Yee Pang , Moore Malcolm A. S.
Published: May 2005

In the present study we investigated the presence of T‐lymphocyte progenitors in the long‐term murine bone marrow culture system described by Dexter: mature Thy‐1 antigen‐bearing T lymphocytes are lost in these cultures after a few days. By culturing nonadherent cells from such cultures in the presence of a supernatant of concanavalin A‐stimulated spleen cells, a source of T‐cell growth factor, we found that Thy‐1 positive blast cells proliferated together with a second population of Thy‐1 negative cells. These two populations of cells have been maintained in long‐term in vitro cultures by passaging the cells in fresh conditioned medium at regular intervals. Moreover, we have been able to establish pure cultures of the Thy‐1‐bearing blast cells after separating them from the non‐T cells using their adherence property to plastic surfaces. Long‐term cultures of T lymphocytes can thus be established from long‐term marrow cultures as well as from the spleen, thymus or fresh bone marrow.
Total and exchangeable calcium in lymphocytes: Effects of PHA and A23187
Authors: Lichtman Andrew H. , Segel George B. , Lichtman Marshall A.
Published: May 2005

Calcium has been suggested as an internal second messenger when lymphocytes are stimulated by mitogens to enter the cell cycle. We have assessed the effect of 2 lymphocyte stimulants, the plant lectin phytohemagglutinin (PHA) and the calcium ionophore A23187, on human lymphocyte nucleic acid synthesis, total cell calcium content, and 4 5Ca labeling. We have used an ultrasensitive method for the measurement of total cell calcium in the same samples used for radiolabeling. Mitogenic concentrations of A23187 (∼ .25 μ mole/liter) caused an increase in both total cell calcium and 4 5Ca labeling. These increases were almost completely blocked by inhibitors of mitochondrial respiration, suggesting that the calcium increment after ionophore treatment was located in the mitochondria. In contrast, total cell calcium was not altered at optimal mitogenic PHA concentrations (0.1 μg/ml and above). However, at the minimum PHA concentrations that caused stimulation (0.025 to 0.1 μg/ml), the dose response of 4 5Ca uptake was very similar to that of DNA sysnthesis. Importantly, we could not stimulate DNA synthesis with PHA without increasing lymphocyte 4 5Ca labeling. Thus, an increase in total cell calcium is not essential for mitogenesis; however, an increase in 4 5Ca exchange is closely associated with the mitogenic effects of A23187 and PHA.
H‐2 I alloantigens and recall of memory cytotoxic responses
Authors: Orosz Charles G. , Macphail Stuart , Bach Fritz H.
Published: May 2005

AQR mice were immunized with H‐2K and H‐2 I encoded alloantigens presented by (Ax6R)F1 splenocytes. Spleen cells from these alloimmune mice were subsequently restimulated in vitro with B10.A lymphocytes and/or B10.T(6R) lymphocytes, thus presenting them with the immunizing H‐2K and H‐2 I alloantigens independently. When stimulated with B10.A lymphocytes, alloimmune lymphocytes develop significant cytotoxicity against the immunizing H‐2K target antigens. When stimulated with a similar number of B10.T(6R) spleen cells, alloimmune lymphocytes undergo a prominant proliferative response, but develop little, if any, cytotoxicity against the immunizing H‐2 K target antigens. The most efficient restimulation of cytotoxicity occurs when the alloimmune spleen cells are simultaneously restimulated by B10.A and B10.T(6R) lymphocytes. Stimulation with the immunizing H‐2 I alloantigens alone is not sufficient for regeneration of detectable cytotoxic responses from alloimmune spleen populations. Stimulation with the immunizing H‐2K alloantigens alone appears to be both necessary and sufficient to stimulate alloimmune cytotoxic responses. Although the immunizing H‐2 I alloantigens are apparently not required to generate alloimmune cytotoxic responses, they markedly potentiate the cytotoxic responses induced by the immunizing H‐2K alloantigens.
An in vitro assay for T lymphocyte progenitors (CFU‐preT)
Authors: Acuff Bonita R. , Cohen J. John
Published: May 2005

Thy‐1.2 negative progenitors give rise to Thy‐1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen‐stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy‐1 positive, peanut agglutinin‐positive, and active in a pre‐T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU‐preT.
Catalytic activities associated with the enzymes II of the bacterial phosphotransferase system
Authors: Saier Milton H.
Published: May 2005

The phosphotransferase system (PTS) in Escherichia coli is a multifunctional, multicomponent enzyme system. Its primary functions deal with carbon source acquisition, while its secondary functions are concerned with the regulation of bacterial physiology. The primary functions of the system include (1) extracellular detection, (2) unidirectional and exchange transmembrane transport, and (3) phosphoenolpyruvate‐dependent and sugar phosphate‐dependent phosphorylation of the sugar substrates of the system. The secondary functions include (1) regulation of the activities of adenylate cyclase and various non‐PTS permeases and (2) regulation of the induced synthesis of several PTS enzymes. Both the primary and secondary functions appear to be elicited by the binding of a sugar substrate to an Enzyme II complex. One of these integral transmembrane enzymes, the mannitol Enzyme II (IImtl), has been solubilized with detergent, purified to homogeneity, and reconstituted in an artificial membrane system. The molecular weight of this protein, IImtl, is 60,000 daltons. It possesses an extracellular sugar binding site and distinct intracellular combining sites for sugar phosphate and phospho‐HPr. An essential sulfhydryl group and an antibody combining site are localized to the cytoplasmic surface of the enzyme, while a dextran combining site is localized to the external surface. Preliminary experiments suggest that the different functions of the Enzyme IImtl can be dissected by genetic and biochemical techniques. These studies emphasize the functional complexity of the PTS and its integral membrane protein constituents.
Interaction of polypeptide neurotoxins with a receptor site associated with voltage‐sensitive sodium channels
Authors: Catterall William A. , Beneski Daniel A.
Published: May 2005

Anthopleurin A, a polypeptide toxin from the Pacific sea anemone Anthopleura xanthogrammica, enhances persistent activation of voltage‐senstive sodium channels by the alkaloid toxins veratridine and batrachotoxin with K0.5 = 20 nM. This effect is inhibited by depolarization. There is a close correlation between enhancement of sodium channel activation and block of [125I] scorpion toxin binding by unlabeled scorpion toxin, sea anemone toxin II from Anemonia sulcata, and Anthopleurin A, indicating that these three polypetide toxins interact with a common receptor site in modifying sodium channel function. Photoactivable derivatives of scorpion toxin label a single Mr ∼ 250,000 polypeptide chain at the polypeptide toxin receptor site. Labeling is blocked by unlabeled scorpion toxin or depolarization and is not observed in variant neuroblastoma clones, which lack sodium channels. These results identify a protein component of the polypeptide toxin receptor site of voltage‐sensitive sodium channels.
Interaction of serum and cell spreading affects the growth of neoplastic and non‐neoplastic fibroblasts
Authors: Tucker R. W. , Butterfield C. E. , Folkman J.
Published: May 2005

Both growth factor availability and cell‐to‐cell contact have been mechanisms used to explain cell growth regulation at high cell density. Recently Folkman and colleagues have shown that changes in cell shape, rather than cell‐to‐cell contact, can regulate the growth of fibroblasts. However, in those studies the relation between serum and shape regulation of growth was not studied, nor were neoplastic and non‐neoplastic cells compared. In this report we have studied these aspects by varying cell spreading and serum concentration independently for 2 non‐neoplastic and 3 neoplastic cell lines. Cell spreading (projected cell area) was controlled by decreasing the adhesiveness of tissue culture plastic plates with poly (hydroxyethyl methacrylate) [poly (HEMA)]. Cell growth was measured as the increase in cell number/day. We have found that more spreading increased net growth of both neoplastic and non‐neoplastic cells, while less spreading (toward rounded configuration) depressed growth. There were also quantitative differences between neoplastic and non‐neoplastic cells. Neoplastic cells continued to grow under conditions of cell rounding, which completely prevented the growth of their non‐neoplastic counterparts. Some neoplastic cells also tended to show little or no increase in net cell number for serum concentrations above 10% as cells became more spread; in contrast, all non‐neoplastic cells grew more with increasing concentrations of serum as they became well spread. Thus, in normal cells, it appears that the sensitivity of cells to humoral factors is governed by cell spreading. This interaction between serum and cell shape is less prominent in some neoplastic cells.
Cleavage of cell surface proteins by thrombin
Authors: Moss Martin , Cunningham Dennis D.
Published: May 2005

This study was based on our previous findings that the mitogenic action of thrombin on cultured fibroblasts can result from interaction of thrombin with the cell surface in the absence of internalization, and that the proteolytic activity of thrombin is required for stimulation of cell division. This prompted us to look for thrombin‐mediated cleavages using 2‐dimensional gel electrophoresis of labeled cell surface proteins. Surface membrane components were labeled by 3 procedures: (1) proteins were labeled by lactoperoxidase‐catalyzed iodination using 125I−; (2) galactose and galactosamine residues of glycoproteins were oxidized with galactose oxidase and reduced with 3H‐NaBH4; and (3) glycoproteins were metabolically labeled by incubating cells with 3H‐fucose. Labeling with the first 2 procedures was carried out after thrombin treatment; in contrast, cells metabolically labeled with 3H‐fucose were subsequently treated with thrombin to look for proteolytic cleavages. Collectively, these studies indicated that only about 5 cell surface proteins were thrombin‐sensitive, consistent with the high specificity of this protease. Each of the labeling procedures revealed a thrombin‐sensitive cell surface glycoprotein which was identified as fibronectin by immunoprecipitation experiments. In addition, cell surface proteins of about 140K and 55K daltons were thrombin‐sensitive. However, cell surface proteins of about 45K daltons and 130K to 1 50K daltons were increased after thrombin treatment. These experiments were conducted on an established line of Chinese hamster lung cells with the eventual goal of studying thrombin‐mediated cleavages of cell surface proteins in a large number of cloned populations derived from this line that are either responsive or unresponsive to the mitogenic action of thrombin. This approach should permit identification of proteolytic cleavages that are necessary for thrombin‐stimulated cell division.
Cell Cycle activation and ouabain‐sensitive ion movements of 3T3 and C3H‐10T½ fibroblasts
Authors: Wenner Charles E. , Cheney John C. , Tomei L. David
Published: May 2005

The introduction of either PGF2α (10−7 M) or TPA (10−7 M) stimulated, ouabain‐sensitive 86Rb+ influx at 30 min in postconfluent 3T3‐4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF2α at these concentrations stimulated the incorporation of 3H‐TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na+/K+ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3‐4 and C3H‐1 0T½ cells there were parallel increases in 3H‐TdR incorporation and ouabain‐sensitive 86Rb+ influxes with 10−7 M TPA, whereas PGF2α stimulated a significant increase in 3H‐TdR incorporation in 3T3‐4 but not C3H‐10T½ cells and only marginal increases in ouabain‐sensitive 86Rb+ influx in both. Therefore, although there appears to be a close correlation between Na+/K+ pump activation and subsequent S‐phase entry following TPA stimulation, a similar correlation for PGF2α cannot be confirmed.
Purification and characterization of multiplication‐stimulating activity (MSA) carrier protein
Authors: Knauer Daniel J. , Wagner Fred W. , Smith Gary L.
Published: May 2005

The rat liver cell line, BRL‐3A, is known to produce a family of polypeptides referred to as multiplication‐stimulating‐activity (MSA). Serum‐free conditioned medium from this cell line is a rich source for the purification of these somatomedin‐like molecules. Somatomedins in serum, as well as MSA produced by BRL‐3A cells in culture, exist primarily as a high molecular weight complex bound to specific carrier proteins. This study describes the purification of the MSA carrier protein (MCP) from conditioned medium using affinity chromatographic procedures. The purified carrier protein is shown to specifically bind labeled MSA and generates a complex with an apparent molecular weight of 60,000–70,000 daltons. Characterization of the carrier protein indicates that it consists of two different noncovalently linked protein chains with apparent molecular weights of 30,000 and 31,500 daltons. The availability of a pure carrier protein should provide a unique opportunity to investigate the functional significance of the carrier protein in the biological activity of the somatomedins.