bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication
Authors: Vintermyr Olav K. , Døskeland Stein Ove
Published: February 2005

Hepatocytes, isolated from adult (250–350 g) rats, attached and survived well in primary culture on highly diluted (< 1 μg/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells “spontaneously” entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca+ + prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar.

The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25–250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.
Possible involvement of reorganization of actin filaments, induced by tumor‐promoting phorbol esters, in changes in colony shape and enhancement of proliferation of cultured epithelial cells
Authors: Sastrodihardjo Sumadhi , Sasaki Yasuto , Shiba Yoshiki , Kanno Yoshinobu
Published: February 2005

Tumor promoters are known to induce reorganization of actin, morphological changes and enhancement of proliferation of epidermal cells in vivo. In this study, we have examined the effects of tumor promoters on these events to clarify the role played by the organization of actin filaments in the regulation of the shape and growth of colonies of epithelial cells in culture. Treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) caused a change in the shape of colonies of FL and Madin‐Darby canine kidney (MDCK) cells within 6 hr. Changes in the shape of colonies were consistent with the morphological change of individual cells and the dissociation of groups of cells in the colonies. Addition of TPA also caused reorganization of actin filaments after 2 hr, and it caused enhancement of proliferation of FL and MDCK cells after 48 hr but did not cause any such changes in KB cells. However, the binding affinities of 4β‐phorbol 12,13‐dibutyrate (PDBu) to FL and MDCK cells were similar to that of PDBu to KB cells. Related tumor promoters such as phorbol 12,13 didecanoate (PDD) and mezerein caused effects similar to those caused by TPA. In contrast, nontumor promoting phorbol esters, such as 4α‐PDD and phorbol, had no effect. Cyclic AMP blocked the TPA‐induced changes in FL and MDCK cells. These results suggest that TPA‐induced reorganization of actin filaments which can be inhibited by cyclic AMP results in changes in the shape of colonies and enhancement of proliferation.
Use of strontium to separate calcium‐dependent pathways for proliferation and differentiation in human keratinocytes
Authors: Praeger Frank C. , Stanulis‐Praeger Betzabe M. , Gilchrest Barbara A.
Published: February 2005

The role of extracellular calcium (Caex) in modulating keratinocyte differentiation has been well documented, but its role in proliferation has been harder to define due to the confounding effect of terminal differentiation. Because strontium (Sr) does not induce terminal differentiation in murine keratinocytes but does mimic the stimulatory effect of Caex on DNA synthesis in chick fibroblasts, experiments were undertaken to determine if Sr could be used to separate the presumably presumably opposing effects of Caex on the proliferation and differentiation of cultured human keratinocytes. In response to additions of SrCl2, keratinocytes in a serum‐free hormone‐supplemented basal medium containing 0.03 mM Ca showed a dose‐dependent increase in day 7 cell yields. Cell yield in the optimal concentration of SrCl2 (1.8 mM) was approximately twice that obtained in any concentration of CaCl2. Maximally stimulatory additions of CaCl2 varied from 0.05 to 1.8 mM, but 0.03 and 0.05 mM additional CaCl2 always increased cell yield relative to unsupplemented controls. Keratinocytes grown in low levels of CaCl2 or any level of SrCl2 have minimal contact with each other regardless of cell density in contrast to the colonies of tightly apposed and stratified cells grown in 1.8 mM CaCl2. Transmission electron micrographs of vertically sectioned confluent cultures in low or high levels of SrCl2 or low levels of CaCl2 revealed abundant ribosomes and keratin filaments but no stratification or desmosomes, while cultures in 1.8 mM CaCl2 were stratified with numerous desmosomes. These results suggest that Caex may separately stimulate keratinocyte proliferation and terminal differentiation and that Srex can substitute for Caex in the former but not the latter process.
α2HS glycoprotein is chemotactic for mononuclear phagocytes
Authors: Malone J. David , Richards Michael
Published: February 2005

α2HS glycoprotein is a normal constituent of plasma. It has a high affinity for hydroxyapatite and is concentrated in bone 100‐fold greater than albumin, however its biological function in bone is not known. We have purified α2HS from human plasma and examined it for evidence of chemotactic activity against human peripheral mononuclear cells, polymorphonuclear leucocytes and lymphocytes in Boyden chamber assays. We observed that the protein was chemotactic for mononuclear cells and that maximal cell migration occured when its concentration in the lower compartment of Boyden chambers was 10−10 M. Chemotaxis did not occur in the absence of a concentration gradient. In addition, cell migration was blocked when the cells were preincubated with 10−10 M α2HS or when the protein was preincubated with rabbit anti‐human α2HS glycoprotein lgG. Neither polymorphonuclear leucocytes, which are responsive to a wide range of chemoattractants, or peripheral lymphocytes exhibited directed migration to α2HS in Boyden chamber assays. The glycoprotein appears therefore to act as a chemotaxin directed to monocyte recruitment, and we speculate that the protein may have an important role in monocyte recruitment to bone and possibly their subsequent fusion and differentiation into functional osteoclasts.
Proliferative potential of human fibroblasts: An inverse dependence on cell size
Authors: Angello John C. , Pendergrass William R. , Norwood Thomas H. , Prothero John
Published: February 2005

Human foreskin fibroblast‐like cells were separated on the basis of DNA content and cell size by fluorescence‐activated cell sorting. Subpopulations of “large” or “small” cells with the same (G1) DNA content were clonally expanded and found to contain predominantly nondividing or highly proliferative cells, respectively. From the rate of clonal growth, we deduce that small cells divide faster than large cells. Intermediate‐sized cells were found to yield primarily smaller (“attenuated”) clones. The clonal data can be incorporated into a previously reported kinetic model of clonal attenuation. This version of the model postulates that small “stem” cells yield larger daughters which have only a limited proliferative potential. We also postulate that a progressive increase in cell size can account for the decreasing concentration of DNA polymerase alpha, which has been reported in older cultures.
Relation of prostaglandin‐induced increases in cellular cAMP to stimulation of pepsinogen secretion from dispersed chief cells
Authors: Raufman Jean‐Pierre , Cosowsky Laurey
Published: February 2005

The actions of prostaglandins (PG) on cAMP in dispersed chief cells from guinea pig stomach were examined and compared to the actions of these agents on pepsinogen secretion. Maximal concentrations of A, B, or E prostaglandins caused a 2–5‐fold increase in pepsinogen secretion and cellular cAMP. The relative order of potency for these actions was PGEs > PGAs > PGBs. Detection of prostaglandin‐induced changes in cAMP was enhanced by adding a phosphodiesterase inhibitor to the incubation solution. The time courses for the effects of prostaglandins on pepsinogen secretion and cAMP were similar. With PGE1 an increase in cAMP and pepsinogen secretion was detected by 1 min and was maximal by 7.5 min. Although significant increases in cAMP were detected with a ten‐fold lower concentration of PGEs than PGAs, a maximal increase in cAMP was observed with the same concentration, 30 μM, of either agent. These data indicate that prostaglandins that stimulate pepsinogen secretion increase cAMP in dispersed chief cells. However, comparison of the dose‐response curves for the actions of prostaglandins on pepsinogen secretion and cAMP revealed that detectable increases in cAMP occurred with concentrations of these agents that were about tenfold greater than those needed to stimulate pepsinogen secretion. Therefore, although the similarity in the kinetics and relative potencies of prostaglandininduced changes in cAMP and enzyme secretion provides further evidence that changes in cAMP play a role in the mediation of prostaglandin‐induced pepsinogen secretion, the present data suggest the involvement of a cellular messenger in addition to cAMP.
Bicarbonate/chloride antiport in vero cells: I. Evidence for both sodium‐linked and sodium‐independent exchange
Authors: Tønnessen Tor Inge , Ludt Jannikke , Sandvig Kirsten , Olsnes Sjur
Published: February 2005

The effect of bicarbonate on the ability of cells to regulate the internal pH after acid and alkali loads was studied. In the presence of Na+, the normalization of the internal pH after acid loads occurred more rapidly in the presence than in the absence of bicarbonate. DIDS (4,4′‐diisothiocyano‐2,2′‐stilbene‐disulfonic acid) strongly inhibited the pH increase, whereas amiloride inhibited it to a lesser extent. The Na+‐linked, bicarbonate‐dependent pHi increase after an acid load was strongly reduced in cells depleted of Cl−. When cells were transferred to gluconate or mannitol balanced buffers containing bicarbonate, there was a rapid alkalinization of the cytosol, apparently due to influx of bicarbonate induced by chloride efflux. When the internal pH was below 7.0, the pH increase was much more rapid in the presence than in the absence of Na+, whereas at higher internal pH, there was no measurable effect of Na+. The ability of the cells to reduce the internal pH after an alkali load was increased in the presence of bicarbonate. The data indicate that both Na+‐linked and Na+‐independent bicarbonate/chloride exchange occur in Vero cells.
Endothelial cells in culture produce a vasoconstrictor substance
Authors: O'Brien Richard F. , Robbins Richard J. , McMurtry Ivan F.
Published: February 2005

We report that cultured vascular endothelial cells release into the culture medium a vasoconstrictor peptide, a substance we call an endotheliumderived constricting factor (EDCF). Conditioned medium from cultured bovine aortic and pulmonary artery endothelial cells caused sustained, dose‐dependent isometric constriction of vascular rings isolated from bovine coronary and pulmonary arteries and rat and guinea pig pulmonary arteries and aortas. The medium also caused vasoconstriction when infused into isolated, perfused rabbit hearts and rat kidneys. Conditioned medium from bovine aortic intimal explants also contained constrictor activity, whereas medium from denuded intimal explants, cultured microvascular endothelial cells, vascular smooth muscle cells, or lung fibroblasts did not. Constrictor activity increased progressively in the culture medium over 2–12 h of incubation. Thrombin stimulated the release of constrictor activity; hypoxia, anoxia and meclofenamate had no effect and the calcium ionophore A23187 inhibited EDCF release. The EDCF caused a characteristic slow‐onset and sustained constriction of the vascular rings that relaxed slowly over 60–90 min following removal. The constriction was not affected by inhibitors of arachidonic acid metabolism or by antagonists of serotonergic, histaminergic, aloha‐adrenergic, opioid, leukotriene, angiotensin II, or substance P receptors; constriction was reversed partly by verapamil and acetylcholine and completely by nitroprusside and isoproterenol. EDCF was heat stable, not extractable into organic solvents, and completely destroyed by trypsin and neutral protease. Cycloheximide blocked the production of EDCF. These properties and the results of polyacrylamide gel filtration experiments suggested that EDCF was a peptide with a molecular weight of 3,000 daltons. These findings show that endothelial cells in culture produce a vasoconstrictor substance and support the idea that endothelial cell products play a role in mediating vascular tone.
Evidence for two functionally different fibrinogen receptors on hemopoietic cells: The glycoprotein IIb–IIIa and the mitogenic fibrinogen receptor
Authors: Levesque Jean‐Pierre , Hatzfeld Antoinette , Hudry‐Clergeon Gilbert , Wilner George D. , Hatzfeld Jacques
Published: February 2005

We have previously established that the mitogenic effect of fibrinogen on hemopoietic cell lines Raji and JM is mediated via a specific receptor (Levesque, J.‐P. et al.: Proc. Natl. Acad. Sci. USA 83:6494–6498, 1986). In this study, we have further characterized the fibrinogen domain involved in the binding to the mitogenic receptor. This binding was not inhibited either by a monoclonal antibody against the C‐terminal sequence of the fibrinogen γchains or by synthetic peptides containing the Arg‐Gly‐Asp sequence. Such inhibition is specific of the platelet fibrinogen receptor, the glycoprotein IIb‐IIIa complex. Fragments containing the fibrinogen D domain were the only plasmin degradation products of fibrinogen which mitogenic. These fragments acted via direct binding on the mitogenic receptor with a Kd of 2.24 × 10−6 M. This value was similar to the K1 value of unlabeled fragments D (2.47 × 10−6 M). Our results suggest the presence of two different functional types of fibrinogen receptors: the glycoprotein IIb–IIIa receptor responsible both for platelet aggregation and leukocyte adhesion and killing, and the mitogenic receptor involved in proliferation control of hemopoietic cells.
Both protein kinase C and calcium mediate activation of the Na+/H+ antiporter in Chinese hamster embryo fibroblasts
Authors: Ober Sharon S. , Pardee Arthur B.
Published: February 2005

Chinese hamster embryo fibroblast cells (CHEF/18) possess a plasma membrane‐associated, amiloride‐sensitive Na+/H+ antiporter that affects intracellular pH (pHi) and is activated by growth factor addition. Our results using 14C‐benzoic acid distribution indicate that both epidermal growth factor (EGF) and thrombin are capable of causing rapid rises in the pHi of CHEF/18 cells. The maximal shift induced by these factors is 0.20 to 0.25 pH units above the basal unstimulated level. Distinctive differences were observed between the modes of action of these two growth factors. Sequential additions revealed that the rise in pHi due to EGF was additive with that caused by diacylglycerols (DAG), while that of thrombin was not. Furthermore, exposure of cells to the phorbol ester PMA for a prolonged period of time in order to down‐regulate protein kinase C (pkC), or treatment with the pkC inhibitor H‐7, abolished the pHi response to thrombin but not to EGF. In contrast, incubation of cells in nominally calcium‐free medium or with the calmodulin antagonists W‐7 or trifluoperazine (TFP) decreased only the ability of EGF to cause changes in pHi. These data suggest that there are two distinct mechanisms for activation of the Na+/H+ antiporter in CHEF/18 fibroblast cells and thus provide an example of the use of alternative modes for the modulation of intracellular processes.
Disparity between expression of transferrin receptor ligand binding and non‐ligand binding domains on human lymphocytes
Authors: Boldt D. H. , Phillips J. L. , Alcantara O.
Published: February 2005

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L‐phytohemagglutinin (LPHA) using two techniques: (1) 125I‐iron‐saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti‐TfR antibodies—OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I‐FeTf per cell, and < 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I‐FeTf binding and antibody binding increased in parallel on LPHA‐activated PBL. After exposure to LPHA for 72 hr, 125I‐FeTf binding increased 100‐fold to 105 molecules per cell and > 50% of cells expressed TfR antigens. By contrast, PMA activation of PBI markedly increased binding of OKT9 and B3/25 but not the binding of 125I‐FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA‐ and PMA‐activated PBL. However, after 72 hr with PMA, 125I‐FeTf binding increased only 6‐fold and consistently remained at < 104 molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down‐regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.
Diminished calcium influx in lectin‐stimulated T cells from old mice
Authors: Miller Richard A. , Jacobson Bruce , Weil Gary , Simons Elizabeth R.
Published: February 2005

T lymphocytes from aged donors function poorly, but the biochemical basis for the defect remains uncertain. We tested the hypothesis that T cells from old mice had a diminished ability to transmit extracellular signals into the cytoplasm, by measuring intracellular free calcium concentrations (Cai) in T cells stimulated by the polyclonal activator concanavalin A (Con A). Using the second‐generation fluorochrome indo‐1 as a reporter of Cai, we found that the Con A‐induced elevation of Cai levels is reduced both in rate and extent in old T cells, as compared to T cells from young mice. Flow cytometric analysis showed that this age‐sensitive change represents a decline, with age, in the number of T cells that can respond to Con A by increasing their Cai above resting baseline levels (100–120 nM). These results thus show that defects in activation are manifested by T cells from old donors within the first 5 minutes of the activation process, and suggest that aging may lead to alterations either in the surface molecules that receive extracellular signals, or in the sequence of coupled events by which these extracellular signals bring about alterations in the intracellular ionic milieu.
Serum‐activated T51B rat liver cells transiently accumulate cyclic AMP‐dependent protein kinases on their surfaces during the G1 phase
Authors: Kleine Leonard P. , Whitfield James F.
Published: February 2005

Confluent T51B rat liver epithelial cells promptly began accumulating cyclic AMP‐binding sites on their surfaces when they were stimulated from quiescence by serum growth factors in medium containing 1.8 mM Ca2+, but they began losing the accumulated binding sites shortly before initiating DNA replication. When the medium contained only 0.02 mM Ca2+, the cells still accumulated surface cyclic AMP‐binding sites, but they did not initiate DNA replication and tended to continue accumulating the binding sites. The cyclic AMP‐binding sites were eliminated completely by treating intact cells for 5 minutes with 0.005% trypsin (which did not damage the cells), and cyclic AMP caused them to be released from intact, undamaged cells into the medium. The binding sites also comigrated electrophoretically with purified regulatory subunits of type I cyclic AMP‐dependent protein kinase, and to a lesser extent the regulatory subunit of type II cyclic AMP‐dependent protein kinase. Therefore, it is likely that a transient accumulation of cyclic AMP‐dependent protein kinases on the outer surface of the plasma membrane is part of the T51B rat liver cell's prereplicate program.
Colony‐stimulating factor‐1 induces thromboplastin activity in murine macrophages and human monocytes
Authors: Lyberg T. , Stanley E. R. , Prydz H.
Published: February 2005

Highly purified colony‐stimulating factor‐1 induced thromboplastin activity in murine macrophages and human monocytes in vitro. The activity increase was inhibited by cycloheximide and prevented by antibodies to CSF‐1.
Human iliac artery endothelial cells express both genes encoding the chains of platelet‐derived growth factor (PDGF) and synthesize PDGF‐like mitogen
Authors: Sitaras N. M. , Sariban E. , Pantazis P. , Zetter B. , Antoniades H. N.
Published: February 2005

In human umbilical vein and bovine aortic endothelial cells in culture c‐sis gene expression and secretion of platelet‐derived growth factor (PDGF) has been previously demonstrated. We now report the presence of PDGF‐1 and PDGF‐2/sis mRNA transcripts in primary cultures of human iliac artery endothelial cells (HIA‐EC). Concomitantly, these cells synthesize and secrete PDGF‐like proteins identified by direct immunoprecipitation with specific PDGF antiserum. The PDGF proteins secreted by HIA‐EC have molecular weights of 31 and 35 kd under nonreducing conditions. Upon reduction these proteins are converted to the monomeric 15‐ and 16‐kd forms. Conditioned media derived from HIA‐EC stimulated the incorporation of 3H‐thymidine by 3T3 cells and competed with 125I‐PDGF for its binding to 3T3 cell membrane receptors. The biologic activity was stable to heating at 100°C for 10 min and sensitive to reducing agents, properties similar to those of authentic PDGF. Production of PDGF‐like mitogen by the human arterial endothelial cells may play an important role in the paracrine modulation of arterial wall regeneration following vascular injury.
Generation of osteoclasts in cultures of rabbit bone marrow and spleen cells
Authors: Fuller K. , Chambers T. J.
Published: February 2005

The primary and specific function of the osteoclast is the resorption of bone. We have applied this criterion, and a monoclonal antibody that binds specifically to osteoclasts, to cultures of tissues that may contain osteoclastic precursors. Bone marrow and spleen cells were incubated for up to 4 weeks in the presence or absence of parathyroid hormone, interleukin 1, or 1,25(OH)2 vitamin D3, on plastic coverslips or slices of devitalised bone. Osteoclasts (as judged by the presence of resorption cavities and the appearance of monoclonal antibody‐positive cells) did not develop in cultures incubated without added hormones, nor in cultures containing parathyroid hormone or interleukin 1, but were regularly observed when bone marrow cells were incubated with 1,25(OH)2vitamin D3. Although multinucleate giant cells were common after incubation, especially in the presence 1,25(OH)2vitamin D3, monoclonal antibody bound not to these cells but to a minor and distinctive population of mononuclear cells and cells of low multinuclearity. We found no excavations and no monoclonal antibody‐positive cells after incubation of peritoneal macrophages with 1,25(OH)2D3. These results provide direct evidence of osteoclastic function arising in cultures of haemopoietic tissues.
Identification of an enhancer‐like element upstream from a cell cycle dependent human H4 histone gene
Authors: Helms S. R. , van Wijnen A. J. , Kroeger P. , Shiels A. , Stewart C. , Hirshman J. , Stein J. L. , Stein G. S.
Published: February 2005

We have identified a segment of DNA in the region 6,500 nucleotides upstream from a cell‐cycle‐dependent human H4 histone gene (pF0108A) which exhibits properties of an enhancer element. This distal element is not required for cap site initiation from the F0108A H4 histone gene. When the enhancer element is present in the genome as a stable integrated sequence, either in its natural upstream location or in a construct where the element is moved just upstream from the proximal promoter sequences, a 25‐fold increase in the level of human H4 histone RNAs is observed. This increased level of mRNA reflects an increase in the rate of transcription. The enhancer effect is also observed when the distal element is inserted in inverse orientation with respect to this gene. In addition, the far upstream element can increase expression of a prokaryotic chloramphenicol acetyl transferase (CAT) gene under control of the simian virus 40 (SV40) early promotor, indicating that the ability to influence transcription is not confined to the gene with which it is normally associated. The ability of the histone gene distal enhancer element to function in both mouse and human cells indicates that transacting regulatory factors encoded by either the human or murine genome are capable of mediating the functional properties of this element, further supporting the cross‐species compatibility of regulatory sequences and molecules that influence transcription of human histone genes.
Cell cycle changes in water properties in sea urchin eggs
Authors: Cameron I. L. , Cook K. R. , Edwards D. , Fullerton G. D. , Schatten G. , Schatten H. , Zimmerman A. M. , Zimmerman S.
Published: February 2005

This study concerned changes in the motional properties of cellular water during the first cell cycle of fertilized sea urchin eggs (Lytechinus variegatus). There was a significant decrease in proton NMR T1 relaxation time and in cytoplasmic ice crystal growth during mitosis and a significant increase in T1 time and cytoplasmic ice crystal size during cleavage. This was not caused by egg water content changes as reflected by egg volume measurements. Removal of both the fertilization membrane and the hyaline layer shortly after fertilization did not alter the pattern of T1 time changes at mitosis and cleavage as compared to whole eggs; thus, the pattern of T1 time changes was attributed to intracellular events. Treatment of fertilized eggs with cytochalasin B, an inhibitor of actin polymerization, did not block the fall in T1 time at mitosis, but did block cytokinesis and the increase in T1 time, which normally occurred at cleavage. A significant pattern of actin disassembly and reassembly at mitosis and cytokinesis was found by studies on the total amount of monomeric actin (G actin) using the DNase I assay. This led to the hypothesis that the observed changes in T1 time and ice crystal size during the first cell cycle were due to the depolymerization and polymerization of cytoplasmic actin. To test this, the effect of the in vitro polymerization of purified actin on the T1 time and on ice crystal growth was examined. It was concluded that changes in the T1 time and ice crystal growth upon polymerization of actin in vitro resembled the changes seen in vivo. These results suggest that changes in the motional properties of cytoplasmic water during the first cell cycle are due, at least in part, to the state of polymerization of cytoplasmic actin.
Covalent modification of the permeability pathways induced in the human erythrocyte membrane by the malarial parasite Plasmodium falciparum
Authors: Breuer W. V. , Kutner S. , Sylphen J. , Ginsburg H. , Cabantchik Z. I.
Published: February 2005

The intraerythrocytic malarial parasite Plasmodium falciparum induces permeation pathways in the plasma membrane of its host, the red blood cell. The pathways display porelike properties with selectivity toward anions and neutral molecules. They are shown here to be susceptible to chemical modification by 4,4′‐diisothiocyano‐2,2′‐dihydrostilbene disulfonic acid (H2DIDS), an amino‐reactive reagent which is impermeant to uninfected cells. At pH 7.4 the reagent affected transport only marginally while freely entering into infected cells and reacting with intracellular hemoglobin. On the other hand, at pH above 8.5, the compound blocked the pathways efficiently (IC50 = 50 μM, at 37°C for 10 min) as judged by four criteria: (1) selective lysis of infected erythrocytes in the presence of isotonic polyols; (2) uptake of [14C] sorbitol into infected cells; (3) uptake of the fluorescent anion Nbd‐taurine into infected cells under conditions in which the native anion transport system was inhibited; and (4) labeling of intracellular hemoglobin by the permeating reagent [3H]H2DIDS. The inhibitory effect was observed only with mature forms of parasitized cells, i.e., from the trophozoite stage and onward, while the pathways of immature ring forms were refractive. However, when the probe was incorporated into the interior of hemoglobin‐depleted resealed ghosts prepared from ring forms, it was found to inhibit the pore‐mediated transport. On the basis of these and other studies we postulate that the H2DIDS‐sensitive sites on the pathways are endofacial, thus requiring penetration of the probe (probably through the same pathway) for their inactivation. Labeling studies with the radiolabeled modifier implicate 120‐Kd, 63‐Kd, and/or 51‐Kd polypeptides as candidates for the pore components.
Infection of L6E9 myoblasts with Trypanosoma cruzi alters adenylate cyclase activity and guanine nucleotide binding proteins
Authors: Morris Stephen A. , Bilezikian John P. , Tanowitz Herbert , Wittner Murray
Published: February 2005

We studied the consequences of infection of L6E9 myoblasts with T. cruzi on the adenylate cyclase complex to test the hypothesis that infection alters the functional properties of the guanine nucleotide regulatory proteins, Ns and Ni. Stimulating activities of adenylate cyclase due to isoproterenol, isoproterenol plus Gpp(NH)p, or forskolin (activities mediated by Ns) are not altered by infection. However, inhibitory activites mediated by Ni [Gpp(NH)p, acetylcholine, and adenosine inhibition of forskolin‐dependent adenylate cyclase activity] are compromised by infection. The reduction in adenosine's inhibition of forskolin‐dependent adenylate cyclase activity is seen throughout the effective concentration range of adenosine. Pertussis toxin does not change basal or stimulated adenylate cyclase activity in infected cells compared with normal uninfected cells, nor does it alter the inhibiting action of adenosine. To evaluate the coupling proteins (Ns and Ni) involved in the stimulation and inhibition of adenylate cyclase more directly, cholera‐ and pertussis‐toxin‐dependent ADP ribosylation studies were performed. The incorporation of [32P]ADP ribose in the presence (specific) or absence (nonspecific) of the toxins was markedly decreased in membranes prepared from infected cells. However, in membranes prepared from infected or uninfected cells previously treated with pertussis toxin, there was a significant reduction in specific pertussis‐toxin‐dependent ADP ribosylation. The infection‐associated diminution in toxin‐dependent ADP ribosylation complements the impaired inhibition of adenylate cyclase data. Collectively, the data further substantiate an infection‐associated alteration in the adenylate cyclase complex, probably at the level of the guanine nucleotide binding proteins.
Rapid in vitro formation of smooth endoplasmic reticulum aggregates within peptide‐producing islet cells
Authors: Knodel Sandra , Meda Paolo , Orci Lelio
Published: February 2005

We report here that heptanol (3.5 mM) induces in vitro a rapid formation of smooth endoplasmic reticulum aggregates (SERA) within isolated islets of Langerhans. SERA appeared after only 15 min of exposure to the alkanol and increased in number during the first 30 min of incubation. At that time, SERA represented 2% and 6% of the volume of B‐ and non‐B‐cells, respectively. Removal of heptanol resulted in the rapid disappearance of SERA, whereas reintroduction of the alkanol rapidly induced these structures again. SERA formation was seen in different types of endocrine and nonendocrine islet cells. In the insulin‐producing B‐cells, SERA formation was not modified by conditions known to alter the secretory activity and the microtubular‐microfilament network or to inhibit protein synthesis. By contrast, SERA formation was inhibted by low temperature and by conditions depleting the energy sources of the cells. Similar observations were made in the presence of either octanol (1 mM) or nonanol (1 mM) but not of shorter chain alkanols, alkanes, oxidative derivates of either heptanol or octanol, and of other unrelated lipid soluble compounds. Incubations in the presence of long‐chain alkanols provide, therefore, a unique model to study in vitro the formation and disposal of smooth endoplasmic reticulum, as well as a system in which rapid membrane biogenesis is amenable to direct experimental testing.
Insulin and α2‐macroglobulin‐methylamine undergo endocytosis by different mechanisms in rat adipocytes: I. Comparison of cell surface events
Authors: Goldberg Ronald I. , Smith Robert M. , Jarett Leonard
Published: February 2005

This ultrastructural study compared the endocytosis of a peptide hormone, ferritin‐labeled insulin (Fm‐I) or gold‐labeled insulin (Au‐I), and a non‐hormonal ligand, gold‐labeled alpha‐2‐macroglobulin‐methylamine (Au‐α2MGMA), by rat adipocytes. Quantitative analysis of the cell surface showed that coated pits occupied 0.4% of the adipocyte surface. This was one fifth to one tenth of that which has been reported on fibroblasts and hepatocytes, cell types in which receptor‐mediated endocytosis has been extensively studied. In contrast, uncoated micropinocytotic invaginations were quite numerous and occupied 13.1% of the adipocyte cell surface. The frequency of microphinocytotic invaginations, 13.8 per μm2 of plasma membrane, was 7–12 times greater than has been reported on fibroblasts. Therefore, the ultrastructure of the endocytic apparatus on rat adipocytes was different from more commonly studied cell types. At 4°C, Au‐α2MGMA concentrated within coated pits to a density that was 52 times greater than that on the uncoated plasma membrane. Au‐α2MGMA was excluded from micropinocytotic invaginations by more than 93%; this exclusion was unrelated to the size of the Au‐α2MGMA particle. In contrast, at 4°C, Fm‐I did not concentrate within coated pits and occupied micropinocytotic invaginations in a random manner. At 37°C, coated pits accounted for all of the endocytosis of Au‐α2MGMA, proving that these structures were functional despite their atypically low density. In contrast, greater than 99% of the endocytosis of Fm‐I or Au‐I occurred through micropinocytotic invaginations. These results demonstrated for the first time by a comparative, quantitative, ultrastructural method that insulin and Au‐α2MGMA undergo endocytosis by dissimilar mechanisms on rat adipocytes. Dissimilarities in the endocytosis of insulin and Au‐α2MGMA may be related to the different biological roles of these two molecules.
Insulin and α2‐macroglobulin‐methylamine undergo endocytosis by different mechanisms in rat adipocytes: II. Comparison of intracellular events
Authors: Goldberg Ronald I. , Smith Robert M. , Jarett Leonard
Published: February 2005

A previous ultrastructural study showed that gold‐labeled insulin (Au‐I) and the non‐hormonal ligand gold‐labeled alpha‐2‐macroglobulin‐methylamine (Au‐α2MGMA) underwent endocytosis by dissimilar cell surface structures on rat adipocytes. The present ultrastructural study compared the intracellular routes taken by these two ligands in adipocytes. Intracellular Au‐α2MGMA was initially found within apparent coated vesicles but Au‐I was not, consistent with the previous demonstration that Au‐α2MGMA underwent endocytosis by coated pits whereas Au‐I was internalized by uncoated micropinocytotic invaginations. Early in the endocytic pathway, the two ligands were segregated within separate small vesicles and tubulovesicles. Au‐α2MGMA was concentrated in a small number of these structures whereas Au‐I was sparsely distributed among a relatively large number. Subsequently, the two endocytic pathways converged as the ligands intermingled within pale multivesicular bodies and lysosome‐like structures. Au‐I was less efficiently transferred to lysosomes than Au‐α2MGMA since a greater proportion of intracellular Au‐I remained associated with small vesicles and tubulovesicles. This study indicates that early intracellular events in the endocytic pathways of insulin and α2MGMA are distinct. These findings are discussed in light of the fundamentally dissimilar biological roles of these two molecules and the possible involvement of the endocytic pathway in the insulin signaling mechanism.
Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis
Authors: Miller Raymond R. , Rao Jasti S. , Festoff Barry W.
Published: February 2005

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle‐specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion‐exchange chromatography on DEAE‐Sephacel followed by gel filtration chromatography on Sepharose CL‐2B. Two cell layer‐associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL‐2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL‐2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle‐derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.
Growth of a human carcinoma (HEp3) in nude mice: Rapid and efficient metastasis
Authors: Ossowski L. , Russo H. , Gartner M. , Wilson E. L.
Published: February 2005

Our aim was to identify conditions which would permit the development of spontaneous metastasis of a human tumor in nude mice in a rapid and predictable manner and to explore ways to quantitate metastasis. Using a human squamous carcinoma–HEp3–we determined that invasiveness and metastasis were influenced by the host. HEp3 cells grew very rapidly and without a significant lag period in Balb/c and NIH(S)‐II nude mice kept in aseptic conditions; a much longer lag period was observed in NIH‐Swiss mice kept in conventional conditions. The HEp3 tumor displayed a highly invasive behavior in N‐NIH(S)‐II mice, in which it invaded the body wall, gaining access to the peritoneal cavity. Microinvasion was noted in all strains of mice inoculated with HEp3 cells. To prolong survival of the mice until metastases became evident, primary tumors were excised when they weighed 1–2 gm. N‐NIH(S)‐II and Balb/c nude mice, maintained in germ‐free conditions, were most receptive to the development of metastases–lung metastases developed in 80% of these mice. Over 60% of all metastases were present within 4 weeks following the removal of the primary. Only 26% of tumor bearing NIH‐Swiss developed lung metastases. Lung metastases were observed in some mice in the absence of local microinvasion, local tumor recurrence, and regardless of the presence of lymph node involvement. They were also noted in mice from which primary tumors were not excised. We compared three methods of lung metastasis detection: histology, detection of tumor cells in the cultures of lung minces, and the measurement of the levels of human urokinase‐type plasminogen activator directly in the lysates of lungs. The detection of tumor cells in cultures of lung minces appeared to be the most sensitive of these methods and the determination of enzyme in lung lysates seemed to hold most promise for a quantitative approach. These data indicate that, the type of tumor, as well as the genetic background and the maintenance conditions of the host, have to be carefully selected to ensure the successful outcome of the particular tumor‐host interaction being studied. Adherence to these guidelines allowed us, in the case of the HEp3 tumor, to develop a rapid, predictable, and efficient model in which to study factors affecting metastasis of this human tumor.
Effects of alterations in endothelial cell volume on transendothelial albumin permeability
Authors: Shepard J. M. , Goderie S. K. , Brzyski N. , Del Vecchio P. J. , Malik A. B. , Kimelberg H. K.
Published: February 2005

We examined the effects of alterations in endothelial cell volume on transendothelial albumin permeability. Studies were done using a confluent monolayer of bovine pulmonary artery endothelial cells grown on gelatinized microporous filters. When endothelial cells were exposed to media made hypertonic with 200 mM mannitol, the intracellular volume (measured with 14C‐urea) decreased twofold and remained decreased over a 30‐minute time‐span, thus showing no significant regulatory volume increase (RVI) within this time period. When endothelial cells were exposed to hypotonic media, intracellular volume rapidly doubled within 2 minutes, and then decreased to baseline values within 10 minutes in spite of the sustained hypotonic environment, a process known as regulatory volume decrease (RVD). We also measured the transendothelial flux of 125l‐albumin with the cells exposed to the same osmotic changes. We observed that only under hypertonic conditions was there a significant change in the 125l‐albumin permeability. These results indicate that the pulmonary artery endothelial cells in culture alter their cell volume when exposed to variations in the osmotic environment, and also show RVD in response to hypotonic conditions but no RVI within 40 minutes after exposure to hypertonic conditions. The transendothelial albumin permeability did not change under hypotonic conditions but increased under hypertonic conditions. Thus, endothelial cells shrinkage may be an important mechanism of increased endothelial macromolecule permeability. These volume changes may occur in endothelial cells in situ and have a role in inducing alterations in the transendothelial permeability to proteins.
Transmembrane electrical characteristics of cultured human skeletal muscle cells
Authors: Iannaccone Susan T. , Li Kai‐Xun , Sperelakis Nicholas
Published: February 2005

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3–4 days, and fusion of myoblasts was observed in 5–10 days. Multinucleated myotubes were impaled under high optical magnification, at 37°C, with conventional glass microelectrodes. The mean resting potential was −44.4 mV ± 2.4 (n = 399); −33 ± 2.3 mV at 9 days (n = 10) vs −48 ± 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M ω (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 ± 2.1 mV with a mean maximum rate of rise (±Vmax) of 72.1 ± 7.5 V/s. The mean overshoot was 13.9 ± 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5–100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.
Changes in erythroid membrane proteins during erythropoietin‐mediated terminal differentiation
Authors: Koury M. J. , Bondurant M. C. , Rana S. S.
Published: February 2005

Membrane and membrane skeleton proteins were examined in erythroid progenitor cells during terminal differentiation. The employed model system of erythroid differentiation was that in which proerythroblasts from mice infected with the anemia‐inducing strain of Friend virus differentiate in vitro in response to erythropoietin (EP). With this system, developmentally homogeneous populations of cells can be examined morphologically and biochemically as they progress from proerythroblasts through enucleated reticulocytes. α and β spectrins, the major proteins of the erythrocyte membrane skeleton, are synthesized in the erythroblasts both before and after EP exposure. At all times large portions of the newly synthesized spectrins exist in and are turned over in the cytoplasm. The remaining newly synthesized spectrin is found in a cellular fraction containing total membranes. Pulsechase experiments show that little of the cytoplasmic spectrins become membrane associated, but that the proportion of newly synthesized spectrin which is membrane associated increases as maturation proceeds. A membrane fraction enriched in plasma membranes has significant differences in the stoichiometry of spectrin accumulation as compared to total cellular membranes. Synthesis of band 3 protein, the anion transporter, is induced only after EP addition to the erythroblasts. All of the newly synthesized band 3 is membrane associated. A two‐dimensional gel survey was conducted of newly synthesized proteins in the plasma membrane enriched fraction of the erythroblasts as differentiation proceeded. A majority of the newly synthesized proteins remains in the same proportion to each other during maturation; however, a few newly synthesized proteins greatly increase following EP induction while others decrease markedly. Of the radiolabeled proteins observed in two dimensional gels, only the spectrins, band 3 and actin become major proteins of the mature erythrocyte membrane. Examination of total proteins of the plasma membrane enriched fractions of EP‐treated erythroblasts using silver staining and 32P autoradiography show that many proteins and phosphoproteins are selectively eliminated from this fraction late in the course of differentiation during the reticulocyte stage. The selective removal of many proteins at the reticulocyte stage of development combined with previous selective synthesis and accumulation of some specific proteins such as α and β spectrin and band 3 in the differentiating erythroblasts lead to the final mammalian erythrocyte membrane structure.