bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha‐fetoprotein‐mediated uptake of fatty acids by human T lymphocytes
Authors: Torres Juan M. , Anel Alberto , Uriel José
Published: February 2005

The binding to resting and activated T lymphocytes of two radiolabelled fatty acids (oleic and arachidonic) was studied in the presence or in the absence of alphafetoprotein (AFP) as carrier protein. Fatty acid binding by resting and activated T lymphocytes was determined at 4°C as a function of the concentration of fatty acid and AFP. Under the conditions employed, the following observations were made: (1) in the presence of AFP, fatty acids (oleic and arachidonic acid) are bound to cells by a two‐component pathway; one is a saturable process, evidenced when the fatty acid to AFP (FA/AFP) molar ratio was fixed at 1 and the concentration of the fatty acid and the protein varied from 0.1 to 3.2 μM, and the second is a nonsaturable function of FA/AFP molar ratio and was linearly related to the unbound fatty acid concentration in the medium over the entire range studied; (2) in the absence of AFP, the nonsaturable process appears to be the only component of fatty acid binding; (3) at all tested concentrations of free (unbound) fatty acid in the medium, net fatty acid binding by either resting or activated T cells was considerably greater in the presence than in the absence of AFP; (4) in the presence of AFP, fatty acid binding was much higher in activated T cells than in resting T cells, whereas in the absence of AFP, nonsignificant differences were observed between activated and resting T cells; and (5) the time course of fatty acid and AFP binding at 4°C revealed that, at equilibrium, the number of fatty acid molecules bound to the cell was much greater than that of AFP suggesting an accelerated dissociation of the fatty acid upon interaction of the AFP‐fatty acid complex with putative cell receptors.

It is concluded to the existence of an AFP/AFP‐receptor pathway that facilitates the binding of fatty acids to T lymphocytes, particularly upon their blast transformation. This pathway may fulfill the increased requirement for fatty acids characteristic of proliferating cells and may serve to regulate the endocytosis of fatty acids with modulatory effects on lymphocyte function and to protect cells from their cytotoxic potential when internalized in excess.
Association of cytoplasmic free Ca2+ gradients with subcellular organelles
Authors: Wahl Miriam , Sleight Richard G. , Gruenstein Eric
Published: February 2005

Previous investigations have identified gradients of intracellular free (Ca2+)i (Ca2+)i in the cytoplasm of human fibroblasts. In this study we have compared the spatial distribution of these gradients with the subcellular distribution of cytoplasmic organelles. Using the Ca2+‐sensitive dye fura‐2 and organelle‐specific fluorescent dyes, we have found that the highest Ca2+ concentrations are found in the perinuclear cytoplasm and that these regions co‐localize with the Golgi apparatus. The area occupied by the endoplasmic reticulum, which includes the Golgi region plus an adjacent area, is also significantly elevated above the average cellular (Ca2+)i. Most mitochondria are located in regions different from those with the highest (Ca2+)i. A variety of phenomena which could have given rise to artifactual (Ca2+)i gradients have been ruled out, including compartmentalization of fura‐2 in subcellular organelles, incomplete hydrolysis of fura‐2AM esters, and the presence of pH gradients which might change the Ca2+ binding characteristics of fura‐2. The existence of gradients in (Ca2+)i between ER and Golgi containing regions of the cytoplasm supports the hypothesis (Sambrook:between Cell 61:197–199, 1990) that the traffic of membrane bound vesicles from ER to Golgi is directed by local variations in (Ca2+)i.
Identification of a new squamous cell differentiation marker and its suppression by retinoids
Authors: Lotan Reuben , Pieniazek Jack , George Margaret D. , Jetten Anton M.
Published: February 2005

Rabbit tracheobronchial epithelial cells (RbTE) can undergo squamous cell differentiation under defined culture conditions and, therefore, have been used as a model to study the regulation of squamous cell differentiation markers. In the present study, we identified a 20‐kDa protein, designated rSQ20, in the serumfree growth medium conditioned by RbTE cells undergoing squamous cell differentiation. The protein was also found in extracts of squamous differentiated cells. rSQ20 was labeled by cells incubated with [35S]methionine but not with [3H]glucosamine, suggesting that it is not a glycoprotein. Undifferentiated cells did not produce this protein. rSQ20 was detected in the conditioned medium of RbTE cells after they reached a confluent and growth‐arrested state, and thereafter its level increased markedly and concurrently with an increase in type I (epidermal) transglutaminase, an established marker of squamous cell differentiation. rSQ20 found in concentrated conditioned medium of squamous differentiated RbTE cells was eluted from a gel filtration column as a protein of 20 kDa, similar to that found by gel electrophoresis under denaturing conditions, suggesting that it is not a multimeric protein. A protein with an apparent molecular weight of 16 kDa (rSQ16), probably the product of partial proteolysis of rSQ20, was often found in various amounts in the conditioned medium of differentiated RbTE cells. β‐Alltransretinoic acid and other vitamin A analogues (retinoids), which suppress squamous cell differentiation, inhibited the expression of rSQ20 in RbTE cells. RbTE cells immortalized by transfection with SV40 large T antigen as well as malignantly transformed derivatives obtained from the immortalized cells by further transfection with v‐Ha‐ras secreted SQ20 and SQ16 when grown to high cell densities although their squamous differentiation was impaired. An analogous protein with an apparent molecular weight of 16 kDa, designated hSQ16, was detected in the medium of differentiated normal human bronchial epithelial (NHBE) cells and normal human epidermal keratinocytes (NHEK). No such protein could be detected in the medium in which undifferentiated NHBE or NHEK cells were grown. These results suggest that rSQ20 and hSQ16 are new markers of squamous cell differentiation. © 1992 Wiley‐Liss, Inc.
Transformation of renal tubule epithelial cells by simian virus‐40 is associated with emergence of Ca2+‐insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers
Authors: Teulon Jacques , Ronco Pierre M. , Geniteau‐Legendre Monique , Baudouin Béatrice , Estrade Simone , Cassingena Roland , Vandewalle Alain
Published: February 2005

We compared the pattern of K+ channels and the mitogenic sensitivity to K+ channel blocking agents in primary cultures of rabbit proximal tubule cells (PC.RC) (Ronco et al., 1990) and two derived SV‐40‐transformed cell lines exhibiting specific functions of proximal (RC.SV1) and more distal (RC.SV2) tubule cells (Vandewalle et al., 1989). First, K+ channel equipment surveyed by the patchclamp technique was modified after SV‐40 transformation in both cell lines; although a high conductance Ca2+‐activated K+ channel [K+200 (Ca2+)] remained the most frequently recorded K+ channel, the transformed state was characterized by emergence of three Ca2+‐insensitive K+ channels (150, 50, and 30 pS), virtually absent from primary culture, contrasting with reduced frequency of two Ca2+‐sensitive K+ channels (80 and 40 pS). Second, quinine (Q), tetraethylammonium ion (TEA) and charybdotoxin (CTX), at concentrations not affecting cell viability, all decreased 3H‐TdR incorporation and cell growth in PC.RC cultures, but only TEA had similar effects in transformed cells. The latter were further characterized by paradoxical effects of Q that induced a marked increase in thymidine incorporation. Q also exerted contrasting effects on channel activity: it inhibited the [K+200 (Ca2+)] when the channel was highly active, with a Ki (0.2 mM) similar to that measured for 3H‐TdR incorporation in PC.RC cells (0.3 mM), but increased the mean current through poorly active channels. TEA blocked all K+ channels with conductance ≥ 50 pS, including the [K+200 (Ca2+)], in a range of concentrations that substantially affected cell proliferation. The unique effect of TEA on SV‐40‐transformed cells might be related to broad inhibition of K+ channels. © 1992 Wiley‐Liss, Inc.
Model for the regulation of platelet volume and responsiveness by the trans‐membrane Na+/K+‐pump
Authors: Marx Gerard , Blankenfeld Avigdor , Panet Rivka , Gorodetsky Raphael
Published: February 2005

The correspondence between K+ uptake in platelets to their responsiveness was studied using 86Rb+ as an analogue of K+. An average 86Rb+ uptake rate of 0.73 (± 0.140) × 10−15 mole Rb+/min‐plt (n = 20) was observed. By the use of K+‐influx inhibitors, we were able to distinguish three distinct 86Rb+ uptake pathways: an ouabain‐sensitive (61% ± 2% inhibitable) pump and two equivalent channels, only one of which is sensitive to furosemide. Other platelet parameters were also examined in conjunction with K+‐uptake. Platelets incubated with ouabain exhibited an overall rise in their cell volume (MPV) with incubation time (ΔMPV = 7.4 × 10−17 L/min 1plt−1). Concomitantly, over 24 hours, a steady decrease in platelet number was recorded by blood cell coulter, which correlated inversely with the counts of particles, which by their size resemble white blood cells (r = 0.89). On a cellular level. incubation with ouabain induced greater expression of surface fibrinogen‐receptor (GPIIb), increased binding of FITC‐labelled fibrinogen, and increased responsiveness to ADP. Our observations suggest the following sequence of events: Ouabain turns off the Na+/K+‐ATPase pump, which leads to water accumulation in platelets and concomitant increased MPV. Greater expression of fibrinogen receptors on the distended platelet surface corresponds to spontaneous microaggregate formation as well as greater respon‐siveness to agonists. Our model links volume regulation, the expression of fibrinogen receptors, and the sensitivity of platelets to agonists to the activity of the Na+K+‐ATPase pump. © 1992 Wiley‐Liss, Inc.
Cytoskeletal events underlying dendrite formation by cultured pigment cells
Authors: Lacour Jean‐Philippe , Gordon Philip R. , Eller Mark , Bhawan Jag , Gilchrest Barbara A.
Published: February 2005

In contrast to neurite outgrowth, pigment cell dendrite formation is relatively unstudied. Keratinocyte‐conditioned medium (KCM) induces a striking dendricity in human melanocytes and B16 melanoma cells that is detectable within 30 min, maximal in 24–48 hr, and quantifiable by computerized image analysis. Cyto‐chalasin B (CB), known to disrupt actin microfilaments, completely blocks dendrite formation if added to cultures before or with KCM. This effect is rapidly reversible, and dendrites appear within 1 hr after refeeding with KCM alone. In contrast, CB treatment fails to disrupt existing dendrites previously induced by KCM. Agents known to cause microtubule disassembly (colchicine, nocodazole, or vinblastine) do not inhibit dendrite formation if added before or with KCM. In contrast, these agents disrupt established dendrites Inhibition of protein synthesis with cycloheximide or actinomycin D completely blocks dendrite formation, but if cultures are provided fresh KCM lacking protein synthesis inhibitors, dendrites reappear within 24 hr. Actin microfilaments visualized with a monoclonal antibody or rhodamine‐phalloidin are poorly organized in untreated cells, but form numerous fibers localized along dendrites in KCM‐treated cells. Microtubules visualized with a monoclonal anti‐tubulin antibody are localized in the center of dendrites. These cytoskeletal changes occur without altering β actin or β tubulin mRNA levels. Taken together, these data implicate actin microfilaments in dendrite outgrowth, but not in maintenance, and conversely microtubules in dendrite maintenance but not in formation. These keratinocyte‐induced changes involving β actin and β tubulin polymerization appear to require both new protein synthesis and post‐translational regulation. The observed similarities between melanocytes and other neural crest‐derived cells suggest that cutaneous pigment cells might serve as an alternative model for studies of neurite outgrowth. © 1992 Wiley‐Liss, Inc.
Interferon‐γ and interleukin‐1β inhibit adipoconversion in cultured rodent preadipocytes
Authors: Grégoire Francine , de Broux Nathalie , Hauser Nadine , Heremans Hubertine , van Damme Jo , Remacle Claude
Published: February 2005

Cytokines like tumor necrosis factor (TNF), interferon‐γ (IFN‐γ), and interleukin‐1 (IL‐1) are known to interfere with the differentiation of cultured cell lines of adipocyte precursors. In the present study, the effect of mouse and rat IFN‐γ, as well as human IL‐1β, was investigated on rodent preadipocytes in primary cultures, either in the presence of fetal bovine serum (FBS, 10%) or in serum‐free defined medium. IFN‐γ exerted an antiproliferative action that was more pronounced when cells reached confluency than during the growth phase of the culture. Morphological observation and quantifications of undifferentiated and differentiating cells revealed that IFN‐γ caused a decrease in the proportion of cells devoid of lipid droplets which would correspond to fibroblast‐like cells, whereas preadipocytes remained unaffected. IFN‐γ induced a marked retardation of adipoconversion, resulting in a partial inhibition of lipoprotein lipase (LPL) activity and a severe decrease in glycerol‐3‐phosphate dehydrogenase (GPDH) activity. The antiproliferative and anti‐LPL effects of IFN‐γ were neutralized by adding anti‐IFN‐γ antibodies, while these antibodies prevented only partially the depressing effect of IFN‐γ on GPDH activity. Contrary to IFN‐γ, IL‐1β slightly enhanced the proliferation in preadipocyte cultures. IL‐1β also depressed adipoconversion, inhibited markedly LPL activity, and partially reduced GPDH activity. These results show that the influence of cytokines on adipoconversion observed in preadipocyte cell lines can be tound in normal preadipocytes in culture. © 1992 Wiley‐Liss, Inc.
Proliferative and differentiative response of corneal and limbal epithelium to extracellular calcium in serum‐free clonal cultures
Authors: Kruse Friedrich E. , Tseng Scheffer C. G.
Published: February 2005

An increasing concentration of extracellular Ca2+ ([Ca2+]e) consistently induces epithelial differentiation, but its effect on proliferation remains variable. We investigated the effect of [Ca2+]e on two different cell populations: the peripheral corneal (PC) and limbal (L) epithelia, the latter containing corneal stem cells. Primary clonal (18 cells/cm2) cultures from rabbit limbal and peripheral corneal epithelia were established in serum‐free MCDB 151 medium containing growth‐promoting agents and 0.03, 0.3, or 1.8 mM Ca2+. During early culture life, colony size and the BrdU labelling‐index of L and PC, assayed on day 6, increased in response to increasing [Ca2+]e; cell attachment and colony‐forming efficiency remained unchanged for both L and PC epithelia. These results indicate that increasing [Ca2+]e, under these defined conditions, stimulates the proliferation of transient amplifying cells, but does not stimulate the differentiation of stem cells into clonal proliferation. A 10‐fold increase of the seeding density or prolongation of the culture up to day 14 or 21 changed the response to [Ca2+]e allowing better proliferation in lower [Ca2+]e. Only cells grown as a monolayer in 0.03 mM Ca2+ could still be passaged on day 14, whereas cells in higher [Ca2+]e showed increasing stratification and cell detachment and could not be passaged. Normal cellular differentiation accessed by the expression of a cornea‐type K3 keratin, recognized by the monoclonal antibody AE‐5, was enhanced by increasing [Ca2+]e. Abnormal differentiation featured by the formation of cornified envelopes was only observed in higher [Ca2+]e. These results indicate that [Ca2+]e promotes the proliferation of relatively undifferentiated transient amplifying cells under clonal, serum‐free culture conditions. Factors that enhance differentiation, such as seeding density or prolonged culture life, can modify this response and allow better proliferation in low [Ca2+]e. © 1992 Wiley‐Liss, Inc.
Characterization of Na+‐K+ homeostasis of cultured human skin fibroblasts in the presence and absence of fetal bovine serum
Authors: Hopp Laszlo , Lasker Norman , Bamforth Ronie , Aviv Abraham
Published: February 2005

Previously, we demonstrated that removal of fetal bovine serum (FBS) from the medium of human skin fibroblasts resulted in an accelerated 86Rb+ washout, decreased cellular K+, and increased Na+ contents. In the present study we examined the mechanism underlying these changes. The efflux rate constant for 86Rb+, and the cellular contents of Na+ and K+ were measured. Verapamil (K1/2= 15 μM) and chlorpromazine (K1/2 = 1 μM) reduced by approximately 70% the increased 86Rb+ washout evoked by FBS removal. The effect of the two drugs was additive at low, but not high, concentrations. Verapamil and chlorpromazine also attenuated the decrease in cellular K+ content and prevented the increase in cellular Na+ content associated with FBS depletion. Bumetanide (50 μM) was only partially effective in offsetting the enhanced 86Rb+ efflux and was completely without any effect on the cellular Na+ and K+ changes induced by FBS removal. In the presence of FBS, A‐23187 produced a slight and transient increase of the 86Rb+ washout. The protein kinase C activator phorbol 12‐myristate 13‐acetate enhanced the 86Rb+ efflux in FBS‐containing medium for a prolonged period but this increase was only a fraction of that caused by serum removal. Cellular Na+ and K+ contents were not changed by the phorbol ester. We conclude that FBS removal raises the cellular Na+ content, and enhances 86Rb+ efflux, through a calmodulin‐dependent pathway activated by calcium influx. © 1992 Wiley‐Liss, Inc.
Mechanism of human keratinocyte migration on fibronectin: Unique roles of RGD site and integrins
Authors: Kim Janice P. , Zhang Ken , Chen John D. , Wynn Kimberly C. , Kramer Randall H. , Woodley David T.
Published: February 2005

The migration of human keratinocytes over the wound bed plays an important role in the re‐epithelialization of cutaneous wounds. Fibronectin, a large glycoprotein matrix component that is abundant within cutaneous wound beds, promotes keratinocyte migration. However, the mechanisms by which keratinocytes migrate over fibronectin are unknown. In this study, we sought to identify specific sites within the fibronectin molecule that induce keratinocyte locomotion and to characterize the cell surface receptors involved. The data show that the domain within the fibronectin molecule that induces human keratinocyte migration is the 120 kD cell‐binding domain close to the carboxyl terminus. The 40 kD heparin‐binding domain near the carboxyl terminus and the 45 kD gelatin‐binding domain near the amino terminus did not promote keratinocyte migration. In addition, keratinocyte migration on both fibronectin and the 120 kD cell‐binding domain was completely inhibited by the presence of GRGDSP peptide, suggesting that keratinocyte migration on fibronectin is mediated by recognizing the RGD sequence located within the cell‐binding domain of fibronectin. Furthermore, keratinocytes were able to migrate directly on immobilized RGD substratum. Cell migration on fibronectin is mediated by the α5β1 integrin since antibodies blocking the α5 and the β1 subunits completely inhibited keratinocyte migration on fibronectin. In addition, we demonstrate that human keratinocytes express α5β1 integrin in culture by flow cytometry. © 1992 Wiley‐Liss, Inc.
Switching from differentiation to growth in hepatocytes: Control by extracellular matrix
Authors: Mooney David , Hansen Linda , Vacanti Joseph , Langer Robert , Farmer Stephen , Ingber Donald
Published: February 2005

Studies were carried out to analyze how different extracellular matrix (ECM) molecules regulate hepatocyte growth and differentiation. Freshly isolated rat hepatocytes were cultured on non‐adhesive plastic dishes that were pre‐coated with defined densities of either laminin, fibronectin, type I collagen, or type IV collagen. Sparse cell plating densities were used to minimize cell‐cell contact formation and all studies were carried out in chemically defined medium that contained a saturating amount of soluble growth factors. Dishes coated with a low ECM density (1 ng/cm2) supported hepatocyte attachment, but did not promote cell spreading or growth. Computerized image analysis confirmed that over 80% of cells remained free of contact with other cells under these conditions. Yet, these round cells maintained high levels of albumin gene expression as well as elevated secretion rates for multiple liver‐specific proteins (albumin, transferrin, and fibrinogen), regardless of the type of ECM molecule used for cell attachment. When ECM coating densities were raised from 1 to 1,000 ng/cm2, cell spreading, expression of histone mRNA, DNA synthesis, and cell proliferation all increased in parallel. Activation of growth by high ECM densities was also accompanied by a concomitant down‐regulation of differentiated functions and again, dishes coated with all four types of ECM molecules produced similar effects. Thus, the ability to switch hepatocytes from differentiation to growth (i.e., between different genetic programs) is not limited to a single ECM molecule, a distinct three dimensional ECM geometry, or due to alteration of cell‐cell interactions. Rather, the regulatory signals conveyed by immobilized ECM molecules depend on the density at which they are presented and thus, on their ability to either prohibit or support cell spreading. © 1992 Wiley‐Liss, Inc.
1,25‐dihydroxy‐vitamin‐D3 enhances antiproliferative effect and transcription of TGF‐β1 on human keratinocytes in culture
Authors: Kim Hee‐Jong , Abdelkader Neamatallah , Katz Marion , McLane John A.
Published: February 2005

Both TGF‐β and 1,25‐dihydroxy‐vitamin‐D3 (1,25(OH)2D3) have been reported to decrease the proliferation of normal human keratinocytes. The effect and expression of TGF‐β in keratinocytes treated with 1,25(OH)2D3 was investigated. Human keratinocytes were grown in the presence of various concentrations of TGF‐β and/or 1,25(OH)2D3 prior to enumeration. TGF‐β, alone, has a half maximal dose of inhibition (ED50) of approximately 750 pg/ml after seven days in culture in Keratinocyte Growth Medium (KGM®; Clonetics) supplemented with 1.5 mM calcium. When 1,25(OH)2D3 (10−7M) was also added to cultures with various concentrations of TGF‐β, the ED50 shifted an average of 2‐fold less. The presence of TGF‐β (10 pg/ml) augmented the potency of 1,25(OH)2D3 by at least 10‐fold. In keratinocyte cultures, the antiproliferative effect of the two compounds together is synergistic. In keratinocytes grown for 1 week in the presence of 1,25(OH)2D3 at 10−6M, the TGF‐β1 message increased approximately 5‐fold. An increase is detected within 2 hours of exposure to 1,25(OH)2D3. There was only a 50% increase in the levels of TGF‐β2 and no detection of TGF‐β3. When keratinocyte cultures were treated with 1,25(OH)2D3 and neutralizing antibodies to TGF‐β, the induced‐antiproliferative activity was blocked by more than 50%. The keratinocytes produced more active than latent TGF‐β after growth with high doses of 1,25(OH)2D3. © 1992 Wiley‐Liss, Inc.
Second messenger signaling in the regulation of cytosolic pH and DNA synthesis by parathyroid hormone (PTH) and PTH‐related peptide in osteoblastic osteosarcoma cells: Role of Na+/H+ exchange
Authors: Sugimoto Toshitsugu , Kano Junichi , Fukase Masaaki , Fujita Takuo
Published: February 2005

The present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH‐related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR‐106 which possessed PTH‐responsive dual signal transduction systems (cAMP‐dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amilorideinhibitable Na+/H+ exchange system. Both human (h)PTH‐(1–34) and hPTHrP‐(1–34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose‐dependent manner with a minimal effective dose of 10−10 M. Dibutyryl cAMP (10−4 M) and Sp‐cAMPS (10−4 M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10−6 M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP‐and Sp‐cAMPS‐induced decrease in pHi but did not affect calcium ionophore‐induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore‐induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA‐inhibited Na+/H+ exchange and increased cytosolic calcium‐induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/H+ exchange system. © 1992 Wiley‐Liss, Inc.
Protein kinase C has both stimulatory and suppressive effects on macrophage superoxide production
Authors: Phillips Wayne A. , Croatto Maryann , Veis Nurin , Hamilton John A.
Published: February 2005

Unlike resident peritoneal macrophages (RPM) or tumor necrosis factor α (TNFα)‐primed bone marrow‐derived macrophages (BMM), unprimed BMM do not generate superoxide in response to the protein kinase C (PKC) activator, phorbol myristate acetate (PMA). However, these cells do contain significant levels of PKC activity. In contrast to PMA, zymosan induces the generation of superoxide in unprimed BMM, as well as in TNFα‐primed BMM and RPM. Staurosporine, a potent PKC inhibitor, failed to affect the zymosan‐induced production of superoxide by unprimed and TNFα‐primed BMM and RPM, in spite of substantial inhibition of PMA‐induced superoxide production by the primed BMM and RPM. However, when PKC was depleted from unprimed BMM by prolonged (24 h) treatment with phorbol dibutyrate (PdBt) (10−7 M) the ability of zymosan to induce the production of superoxide was greatly diminished. Such a result could be interpreted as suggesting a role for PKC in the zymosan‐induced response a conclusion which contrasts with the inhibitor data. However, PKC depletion, in this case, is achieved via the PdBt‐induced activation of PKC. It is thus possible that it is the initial activation of PKC, rather than its depletion, that suppresses superoxide production. Consistent with this interpreation, the co‐stimulation of unprimed BMM with both zymosan and PMA resulted in a reduced superoxide release compared to zymosan alone. The activation of PKC therefore appears to have a suppressive effect on the generation of superoxide by unprimed cells. We thus conclude that PKC is not required for zymosan‐induced superoxide production by either primed or unprimed macrophages and suggest that PKC may be involved in regulatory mechanisms restricting superoxide production by macrophages. However, since PMA alone can initiate the release of superoxide from primed BMM and RPM, it would appear that PKC can mediate both stimulatory and suppressive signals for macrophage superoxide production. © 1992 Wiley‐Liss, Inc.
Imaging neutrophil activation: Analysis of the translocation and utilization of NAD(P)H‐associated autofluorescence during antibody‐dependent target oxidation
Authors: Liang Bing , Petty Howard R.
Published: February 2005

Fluorescence intesified/enhanced microscopy has been used to study the metabolic activation of living human neutrophils in time‐lapse sequences. The autofluorescence associated with NAD(P)H's emission band was studied within individual quiescent and stimulated cells. Excitation of NAD(P)H‐associated autofluorescence was provided by a high‐intensity Hg‐vapor lamp. The background‐subtracted autofluorescence signals were computer enhanced. In some cases the ratio image of NAD(P)H‐associated autofluorescence to tetramethylrhodamine methyl ester (TRME) fluorescence, which was found to be uniformly distributed within neutrophils, was calculated to normalize autofluorescence intensities for cell thickness. Activation of the NADPH oxidase by phorbol myristate acetate, F−, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), or tumor necrosis factor (TNF) dramatically reduced autofluorescence levels. Membrane solubilization with sodium dodecyl sulfate eliminated autofluorescence. Thus, control experiments indicated that most or all of the detectable NAD(P)H‐associated autofluorescence was due to NAD(P)H, consistent with previous non‐microscopic studies. To understand the metabolic events surrounding the internalization and oxidative destruction of targets, we have imaged the NAD(P)H‐associated autofluorescence of neutrophils and the Soret band of antibody coated target erythrocytes during cell‐mediated cytotoxicity. Absorption contrast microscopy of the erythrocyte's Soret band is an especially sensitive indicator of the entry of reactive oxygen metabolites into this target's cytosol. Thus, it is possible to spectroscopically dissect and image the substrate (NADPH) and product (O2−) reactions of the NADPH oxidase in living unlabeled neutrophils. During real‐time experiments at 37°C, the level of NAD(P)H‐associated autofluorescence surrounding phagosomes greatly increases before the disappearance of the target's Soret band. NAD(P)H‐associated autofluorescence in the vicinity of phagocytosed erythrocytes is greatly diminished after target oxidation. This suggests that NAD(P)H is translocated to the vicinity of phagosomes prior to the oxidation of targets. The apparent cytosolic redistribution of NAD(P)H was confirmed by ratio imaging microscopy to control for cell thickness. We suggest that NADPH including its sources and/or carriers accumulate near phagosomes prior to target oxidation and that local NADPH molecules are consumed during target oxidation. © 1992 Wiley‐Liss, Inc.
K‐current mediation of prolactin‐induced proliferation of malignant (Nb2) lymphocytes
Authors: Wang Yu‐Fen , Jia Hongjun , Walker Ameae M. , Cukierman Samuel
Published: February 2005

The effects of different concentrations of various K‐current blockers on prolactin‐induced proliferation and membrane K‐currents in malignant lymphocytes (Nb2 cells) were investigated. Membrane currents were measured with the whole cell patch‐clamp technique, and lymphocyte density was quantified by both spectrophotometric and conventional methods. K‐current blockers tested (quinidine, 4‐aminopyridine, barium, and tetraethylammonium) exhibited similar rank order potency for K‐current block and inhibition of prolactin‐induced proliferation of malignant lymphocytes. Because Nb2 cells proliferate independently of a transmembrane Ca‐influx, these results suggest that K‐currents per se rather than K‐current modulation of Ca‐influx is an essential event for lymphocyte proliferation. © 1992 Wiley‐Liss, Inc.
Production of granulocyte colony‐stimulating factor and granulocyte/macrophage‐colony‐stimulating factor after interleukin‐1 stimulation of marrow stromal cell cultures from normal or aplastic anemia donors
Authors: Migliaccio Anna Rita , Migliaccio Giovanni , Adamson John W. , Torok‐Storb Beverly
Published: February 2005

We have studied stromal cell function in naive or interleukin‐1 (IL‐1)‐stimulated (100 pg/ml) long‐term marrow cultures (LTC) from 12 normal donors and 21 patients with severe aplastic anemia (AA). Conditioned media (CM) from normal LTC contained levels of erythroid burst‐promoting activity (BPA) and granulocyte/macrophage (GM) colony‐stimulating activity (CSA) comparable to those previously described (Migliaccio et al., [1990] Blood, 75:305–312). The addition of IL‐1 to these cultures increased the level of CSA and, specifically, of granulocyte colony‐stimulating factor (G‐CSF) released. Anti‐GM‐CSF antibody neutralized BPA and CSA in normal naive LTC CM but only the CSA in the CM from IL‐1‐stimulated LTC. Since the concentrations of GM‐CSF, as detected with a specific immunoassay, did not increase after IL‐1 treatment, these data suggest that IL‐1‐stimulated cultures contain an unidentified growth factor having BPA. CM from AA stromal cells contained levels of CSA comparable to those observed in normal stromal cell CM but had significantly lower levels of BPA. Neither anti‐GM‐CSF nor anti‐IL‐3 antibodies neutralized the BPA in AA stromal cell CM. This activity may be related to that found in the CM of IL‐1‐treated normal stromal cells. In nearly 50% of stromal cell cultures of AA patients, addition of IL‐1 failed to increase the BPA, CSA, or G‐CSF. The presence of an inhibitor in naive or IL‐1‐treated AA stromal cell CM was excluded by adding the CM to IL‐3‐stimulated cultures. These findings suggest that G‐CSF and GM‐CSF genes are differentially regulated in the marrow microenvironment. Furthermore, a marrow microenvironment, deficient in BPA production and, in some cases, unresponsive to IL‐1 could contribute to marrow failure in some patients with AA. © 1992 Wiley‐Liss, Inc.
Effect of hypoxia upon intracellular calcium concentration of human endothelial cells
Authors: Arnould Thierry , Michiels Carine , Alexandre Isabelle , Remacle José
Published: February 2005

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2.

We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (β‐hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia‐induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i.

These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation. © 1992 Wiley‐Liss, Inc.
Modulation of mRNA levels during human keratinocyte differentiation
Authors: Younus Jawaid , Gilchrest Barbara A.
Published: February 2005

Cultures of human keratinocytes provide an excellent model system in which to study differentiation. Using the phorbol ester 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) and calcium, two agents known to induce keratinocyte differentiation in vitro, we examined the expression of the genes encoding c‐fos, c‐myc, and c‐jun; involucrin, a protein precursor of the keratinocyte cornified envelope; and L‐7, a ribosomal protein. Overall, at the doses studied, TPA induced a more rapid and profound differentiation than did calcium, as evaluated by culture morphology and northern blot analysis. Our studies showed a constant low level of c‐fos and c‐jun expression in unstimulated cells with no significant change after addition of either TPA or calcium except when transcript breakdown was inhibited by cycloheximide. The c‐myc proto‐oncogene, known to have a high constitutive expression in actively proliferating cells, was strongly downregulated by TPA, but calcium had no effect over a 32 hour period, consistent with the greater growth inhibition of TPA in this system. Involucrin was induced about ninefold by both TPA and calcium as early as 8 hours after stimulation, suggesting transcriptional regulation of this gene during differentiation. L‐7, recently demonstrated to be downregulated in late passage human fibroblasts in an in vitro model of senescence, was also strongly downregulated by either TPA or calcium, consistent with an interrelationship between the basic cellular processes of aging and differentiation. These finding expand our knowledge of the differentiation process in human keratinocytes. © 1992 Wiley‐Liss, Inc.
Cytokines involved in monocyte mediated tumor cell death and growth inhibition in serum‐free medium
Authors: Van Der Bosch Jürgen , Rüller Elisabeth , Ernst Martin , Schade Ulrich F. , Mathison John C. , Rüller Stephan , Schlaak Max
Published: February 2005

In a serum‐free culture system, the release of TNF, II‐1, II‐6, IFN‐α, and IFN‐β during interaction of elutriated human monocytes (MO) with human tumor cells (TC) was studied by ELISA‐technique. Contributions of these cytokines to inhibition of TC‐growth and to induction of TC‐death by supernatants (SU) gained from such MO/TC‐interaction cultures were investigated using affinity chromatography for removal of individual cytokines. Although the TC used are relatively insensitive to recombinant human TNF, withdrawal of TNF causes 50% to 75% reduction of SU‐induced TC‐death rates, suggesting that susceptibility to TNF is raised during MO/TC‐interaction by the other cytokines. Individual removal of other cytokines does not cause reduction of SU‐mediated TC‐death. However, combined withdrawal of II‐1 and IFN‐α/β causes in 2 of 4 TC‐lines significant reduction of TC‐death. Combined removal of TNF, IFN‐α/β, II‐1, and II‐6 leads to complete prevention of SU‐mediated growth inhibitory and lytic effects, suggesting that besides these cytokines other signals are not involved significantly. SU‐effects can be mimicked by appropriate combinations of authentic cytokines. The response of TC to SU‐or cytokine‐exposure is strikingly dependent on TC‐density, leading at subconfluent TC‐density exclusively to inhibition of growth and at postconfluent TC‐density to induction of cell death. The principal effect of SU or cytokine combinations in this context seems to be the activation of growth inhibitory signal transduction pathways leading to TC‐death in postconfluent TC‐populations exclusively if growth stimulatory pathways are activated at the same time. Mouse L cells do not follow this reaction pattern: Their death is exclusively dependent on the presence of TNF in SU and they die upon SU‐exposure at postconfluent as well as at subconfluent cell density. © 1992 Wiley‐Liss, Inc.
TGF‐beta inhibits proliferation of and promotes differentiation of human promonocytic leukemia cells
Authors: Bombara Christine , Ignotz Ronald A.
Published: February 2005

Transforming growth factor‐beta 1 (TGF‐β1) has been implicated in a variety of responses associated with wound healing and inflammation. Thus, TGF‐β1 enhances production of several extracellular matrix proteins both in vitro and in vivo, is chemotactic for monocytes, and alters the functioning of lymphocytes. We have examined the ability of TGF‐β1 to affect the behavior of human THP‐1 promonocytic leukemia cells, a cell line with the capacity to differentiate into macrophage‐like cells. TGF‐β1 reduces the growth rate of these cells, induces morphologic changes, and promotes adherence to culture surfaces. In addition, the adherent cell population expresses high levels of esterase activity, acquires the ability to ingest latex beads, and releases elevated levels of interleukin 1. TGF‐β1‐treated cells also express elevated levels of the β2 family of integrins. Taken together, these results suggest that TGF‐β1 is capable of promoting the maturation of promonocytic cells into macrophages. This outcome has implications at wound sites where TGF‐β1 and a myriad of other factors interact with many cell types to facilitate healing. © 1992 Wiley‐Liss, Inc.
Phorbol ester‐induced promyelocytic leukemia cell adhesion to marrow stromal cells involves fibronectin specific α5β1 integrin receptors
Authors: Martin‐Thouvenin V. , Gendron M. C. , Hogervorst F. , Figdor C. G. , Lanotte M.
Published: February 2005

The human promyelocytic cell line NB4 exhibited a weak adhesion capacity in bone marrow‐derived stromal cells and their extracellular matrices (5–15% of adherent cells). Adhesion was enhanced by pulse‐treatment of cells with phorbolester (PMA 10−7 M). Adhesion was induced within minutes, was fibronectin‐specific, and affected up to 100% of the treated cells. This biological response to PMA resulted from the activation of protein kinase C (PKC), since PKC inhibitors (staurosporine, sphingosine, CGP 41251, and calphostin C) prevented the phenomenon. Phenotypical analysis of integrin receptor expression (particularly FN receptors VLA‐4 and VLA‐5) at the membrane of untreated or PMA‐treated cells revealed that PMA induced no significant modification of the level of expression of these receptors. However, inhibition studies carried out with anti‐VLA monoclonal antibodies demonstrated that the FN‐specific adhesion triggered by PKC involved the α5β1 FN‐specific receptors (VLA‐5) We showed that the bindings of NB4 cells to fibronectin was RGD‐dependent. PMA‐induced adhesion was not correlated to phosphorylation of the VLA‐5 receptor. These findings may partially explain the malignant behaviour of these cells: The loss of their capacity to adhere to stomal cells may arrest differentiation and explain the large number of leukemic cells in the circulation. © 1992 Wiley‐Liss, Inc.
Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells
Authors: Li Shiwei , Plowman Gregory D. , Buckley Sharon D. , Shipley Gary D.
Published: February 2005

Normal human mammary epithelial cells (HMECs) proliferate in a serum‐free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin‐regulated, EGF‐like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrin growth factor to support the EGF‐independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL‐100) and six mammary tumor‐derived cell lines had low to undetectable levels of AR. The EGF‐indendent growth of HMECs was blocked by the addition of heparin or a monoclonal anti‐RGF receptors antibody to the culture medium, implication AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor‐derived cell line, Hs578T, which proliferates in an EGF‐independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type‐alpha (TGF‐α) mRNA revealed coordinate expression of AR and TGF‐α in these cells. These data suggest that both AR and TGF‐α mRNA are produced in much greater abundance by normal HMECs than in tumor‐derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs. © 1992 Wiley‐Liss, Inc.
Plasminogen activator inhibitor‐1 is induced in migrating endothelial cells
Authors: Pepper M. S. , Sappino A. P. , Montesano R. , Orci L. , Vassalli J.‐D.
Published: February 2005

It has been proposed that a finely tuned protease‐anti‐protease equilibrium must be mainted during process of cell migration in order to limit extracellular proteolysis to the close proxmity of the cell surface, and therby to prevent excessive extracellular matrix degradation. We have previously shown that urokinase‐type plasminogen activator (u‐PA) activity is induced in microvascular endothelial cells migrating from the edges of a wounded monolayer in vitro (Pepper et al., J. Cell Biol., 105:2535–2541, 1987). By Northern analysis, we now demonstrate that plasminogen activator inhibitor 1 (Pal‐1) mRNA is increased in multiple‐wounded monolayers of bovine microvascular (BME) or aortic (BAE) endothelial cells, with a maximal 7‐ and 9‐fold increase 4 h after wounding, respectively. By in situ hybridization, we demonstrate that the increase in PAI‐1 mRNA in localized to cells at the edge of a wounded BME or BAE cell monolayer. The increase in PAI‐1 mRNA observe in BME cells is independent of cell division and is inhibited by antibodies to basic fibroblast growth factor (bFGF), suggesting that PAI‐1 induction in migrating cells is mediated by the autocrine activity of bFGF. Taken together with our previous observations on the induction of u‐PA, these results support the hypothesis that the proteolytic balance in the pericellular environment of migrating cells is regulated through the concomitant production of proteases and protease inhibitors. © 1992 Wiley‐Liss, Inc.
Monoclonal antibody that recognizes the carbohydrate portion of cell adhesion molecule L1 influences calcium current in cultured neurons
Authors: Asou Hiroaki
Published: February 2005

A monoclonal antibody (mAb), 2E12, against the neural cell adhesion molecule L1 recognized the 200 kDa component of L1. The epitope of L1 reacting with mAb 2E12 was localized in its carbohydrate chain, judging from the results of experiments on glycopeptidase F treatment. The physiological effect of adding mAbL1 (2E12) to cultured mouse dorsal root ganglion neurons was studied using patch‐clamp techniques. The binding of mAbL1 (2E12) to the neurons expressing L1 molecule induced an inward current inhibited by calcium channel blockers such as nifedipine and Lanthanum. It was also found that the mAbL1 (2E12) leads to a rise in the intracellular Ca2+ concentration ([Ca2+]i) in cultured neurons. This rise seems to be due to an influx of extracellular Ca2+, since treatment with EGTA abolished those phenomena. L‐type calcium channel blockers such as nifedipine and cadmium, as well as inward current, blocked the effect of mAbL1 (2E12). These results suggest that the carbohydrate chain of L1 glycoprotein is directly involved in the induction of calcium current, and that the L1 molecule may play a prominent role in regulation of the Ca2+ channel. © 1992 Wiley‐Liss, Inc.