bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of 3′‐azido‐3′‐deoxythymidine inhibition of ricin and pseudomonas exotoxin A toxicity in CHO and vero cells
Authors: Wellner Robert B. , Pless Dorothy D. , Thompson William L.
Published: February 2005

Ricin (RIC), modeccin (MOD), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are protein toxins that enter cells by receptor‐mediated endocytosis. After intracellular transport and membrane translocation to the cytosol, these toxins inhibit protein synthesis by enzymatically removing a specific adenine residue from ribosomal RNA (RIC, MOD), or by ADP‐ribosylation of elongation factor‐2 (PE, DT). Recently, Thompson and Pace (1992) reported that AZT (3′‐azido‐3′‐deoxythymidine) inhibited RIC toxicity in Vero cells, and this inhibition was not due to a block of RIC enzymatic activity. This paper extends these findings and examines the effects of AZT treatment on the toxicities of other protein toxins in Chinese hamster ovary (CHO) and Vero cell lines. AZT treatment did not significantly alter the toxicity of DT or MOD in either cell line, but it markedly reduced RIC and PE toxicity in both cell lines. The ID50 values (concentration of toxin required to inhibit protein synthesis by 50%) for RIC and PE in CHO cells increased approximately 6.5‐ and 12.5‐fold, respectively; while in Vero cells the ID50 values increased ca. 8.5‐and 4.5‐fold, respectively. Results of further studies revealed differences in the mechanisms by which AZT inhibited RIC and PE toxicity. Results of cell‐free translation indicated that, unlike its effects on RIC, AZT blocked the ability of PE to perform its enzymatic activity. As AZT did not block RIC enzymatic activity, we examined the effects of AZT on earlier steps in the RIC intoxication process. AZT treatment did not inhibit cell‐surface binding or internalization of [125I]‐RIC. Results of kinetic studies showed that when AZT was incubated with cells at the time of RIC exposure, it caused no major change in the lag phase, during which RIC reaches the site of translocation. However, it clearly reduced the subsequent first‐order reduction in the rate of protein synthesis, suggesting an effect on translocation. Monensin (an ionophore that perturbs intracellular trafficking and increases the toxicities of RIC and PE) reduced AZT protection against both toxins. Nocodazole and colchicine (agents that disrupt microtubules and some routes of intracellular trafficking) reduced the ability of AZT to inhibit RIC, but not PE, toxicity. In summary, our results suggest that (1) AZT acts within the cytosol to inhibit (directly or indirectly) the enzymatic action of PE, and (2) the AZT inhibition of RIC cytotoxicity does not involve perturbations of RIC cell‐surface binding, internalization, or enzymatic activity but might result from an alteration in RIC translocation. © 1994 wiley‐Liss, Inc.
Thapsigargin‐induced calcium entry in FRTL‐5 cells: Possible dependence on phospholipase A2 activation
Authors: Törnquist Kid , Ekokoski Elina , Forss Lisbeth
Published: February 2005

Stimulating rat thyroid FRTL‐5 cells with agonists that activate the inositol phosphate cascade results in the release of sequestered calcium and influx of extracellular calcium. In addition, phospholipase A2 (PLA2) is activated. Since PLA2 is a calcium‐dependent enzyme we wanted to investigate the interrelationships between PLA2 activity and the entry of calcium. Stimulating 3H‐arachidonic acid (3H‐AA)‐labelled cells with thapsigargin resulted in a substantial release of 3H‐AA. This release was totally abolished in a calcium‐free buffer. Pretreatment of Fura 2 loaded cells with 4‐bromophenacyl bromide, an inhibitor of PLA2 activity, decreased the thapsigargin‐induced entry of calcium, suggesting a role for PLA2 in the regulation of calcium entry. In cells treated with nordihydroguaiaretic acid (NDGA), clotramizole, or econazole, compounds with lipoxygenase and cytochrome P‐450 inhibitory actions, the thapsigargin‐induced entry of calcium was decreased in a dose‐dependent manner. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the thapsigargin‐induced calcium entry. We also showed that stimulation of the cells with arachidonic acid released sequestered calcium, apparently from the same intracellular pool as did thapsigargin. The results suggested that the calcium‐induced PLA2 activation and the metabolism of the produced arachidonic acid by a noncyclooxygenase pathway may be of importance in maintaining calcium entry after releasing sequestered Ca2+ in FRTL‐5 cells. © 1994 Wiley‐Liss, Inc.
Recovery of nuclear matrix ultrastructure of interphase CHO cells after heat shock
Authors: Wachsberger Phyllis R. , Coss Ronald A.
Published: February 2005

Heat shock induces changes in G1 CHO cell nuclear matrix (NM) ultrastructure that may be related to heat‐induced nuclear protein accumulation (Wachsberger and Coss, 1993, J. Cell. Physiol., 155:615–634). The present study quantitates recovery of alterations in NM fine structure in CHO cells heated in G1 and compares structural recovery with recovery of bulk RNA synthesis and surviving fraction (SF). Morphology of NM preparations was quantified 30 min and 20 hr following heat shock by 1) measurement of the number of fiber anastomosing points per unit area per NM, and 2) measurement of the length of fibers between points of anastomoses within individual NMs. Architectural recovery was nearly complete within 20 hr in cells heated at 43°C or 45°C with SFs of 0.27 or greater. No recovery of architecture was observed in heated cells with SFs of approximately 0.01 or less. The residual damage to NMs was associated with RNA‐containing fiber networks as determined by means of RNase gold labeling. Recovery from inhibition of RNA synthesis following heat shock was related to recovery of NM architecture. It is suggested that 1) repair of NM architecture does not require full recovery of bulk RNA synthesis, and 2) partial or complete irreversible collapse of the NM may be responsible, in part, for heat‐induced, interphase cell death. © 1994 Wiley‐Liss, Inc.
SR 25989 inhibits healing of a mechanical wound of confluent human saphenous vein endothelial cells which is modulated by standard heparin and growth factors
Authors: Klein‐Soyer C. , Cazenave J.‐P. , Herbert J.‐M. , Maffrand J.‐P.
Published: February 2005

The thienopyridine, ticlopidine, a potent platelet antiaggregating agent and SR 25989, an esterified derivative of ticlopidine, devoid of antiplatelet activity, were tested in an in vitro model of healing of a mechanical wound in confluent endothelium. This model allows exploration of substances involved in wound healing and angiogenesis. These two compounds inhibited both cell proliferation and cell migration during lesion repair in a dose‐dependent manner (18–150 μM), SR 25989 being twice as active as ticlopidine. Its effect was not inhibited by acidic or basic fibroblast growth factor or by platelet derived growth factor. In contrast, it exerted a conjugated inhibition with standard heparin and was able to totally reverse the healing increase induced by a mixture of acidic fibroblast growth factor and heparin. The mechanism of action of SR 25989 is not yet elucidated, but it does not seem to involve competition with fibroblast growth factors since these substances were not able to alter their binding to receptors on the endothelial cell surface. SR 25989 therefore appears as a promising new candidate for inhibition of angiogenesis. © 1994 Wiley‐Liss, Inc.
Role for autocrine TGF‐β1 in regulating differentiation of a human leukemic cell line toward osteoclast‐like cells
Authors: Fiorelli G. , Gori F. , Masi L. , Frediani U. , Brandi M. L. , Tanini A. , Bernabei P. A. , Ballock R. T. , Wakefield L. M. , Sporn M. B.
Published: February 2005

Increasing evidence suggests that transforming growth factor‐β (TGF‐β) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF‐β1 and that exogenous or autocrine TGF‐β1 can induce the same features of osteoclastic‐like cells, exerting its effects through the binding to TGF‐β specific receptors. Scatchard analysis of 125I‐labeled TGF‐β1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of ∼25 pM and a binding capacity of ∼900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF‐β receptors. Stimulation of FLG 29.1 cells with low TGF‐β1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF‐β1 caused a significant and dose‐dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF‐β1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF‐β1 mRNA expression and growth factor release. The majority of TGF‐β1 secreted by TPA‐treated cells was in its latent form. However, anti‐TGF‐β antibodies inhibited TGF‐β1 and TPA‐induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF‐β1 and indicating that the cells can activate and respond to the TGF‐β that they secrete. These findings support a potential autocrine role for TGF‐β1 in osteoclast differentiation. © 1994 Wiley‐Liss, Inc.
Mouse fibroblasts defective in thrombin mitogenesis possess functional proteolytically activated receptor for thrombin: Requirement for a second signaling pathway
Authors: Kim Dennis W. , Wang Fang , Ramakrishnan Shyam , Scott David L. , Hensler Theresa M. , Thompson William C. , Carney Darrell H.
Published: February 2005

Thrombin mitogenesis in fibroblasts requires two distinguishable subsets of signals; one generated by proteolytic cleavage, the other by high‐affinity cell surface binding. Characterizing two closely related mouse embryo (ME) cell lines with high numbers of thrombin binding sites, we found that one line, B11‐A, responds mitogenically to thrombin, epidermal growth factor (EGF), and serum, whereas the B11‐B cell line is responsive to EGF and serum, but not to thrombin. The B11‐B defect responsible for loss of thrombin responsiveness is not due to differences in the number of high‐affinity binding sites, the affinity of thrombin binding to these sites, or to differences in cell surface expression of proteolytically activated receptors for thrombin (PART). The defect is also not associated with an inability of thrombin to activate PART since thrombin stimulates the cleavage‐dependent induction of the proto‐oncogene c‐fos in both B11‐A and B11‐B cells. Various combinations of thrombin, synthetic thrombin receptor peptide, TRP‐14 (SFFLRNPGENTFEL), platelet‐derived growth factor (PDGF), and phorbol 12‐myristate 13‐acetate (PMA) were used to better define the defect in thrombin‐mediated mitogenesis in B11‐B cells. Direct activation of protein kinase C with PMA in combination with thrombin did not overcome B11‐B nonresponsiveness. However, mitogenic responsiveness was regained in B11‐B cells by simultaneous addition of PDGF and either thrombin or TRP‐14. Therefore, the B11‐B defect may involve a set of signals initiated by nonproteolytic thrombin interactions distinct from those initiated by PART, but related to the downstream signals initiated by the tyrosine kinase‐associated growth factors, EGF and PDGF. © 1994 Wiley‐Liss, Inc.
Apoptotic cell death induced by serum and its prevention by thiols
Authors: Kurita Takeshi , Namiki Hideo
Published: February 2005

We recently reported that serum contains low molecular weight factors that inhibit growth and cause cell death in vitro. The present study focused on identifying components of basal media that counteract the toxic effects of serum. Amino acids L‐cyst(e)ine and L‐tryptophan were found to prevent serum‐induced cell death of TIG‐1 human fetal lung fibroblasts and other cell types. In addition to L‐cysteine, other thiol‐bearing and dithiol‐cleaving compounds showed a similar ability to rescue the cells. Various inhibitors of protein or RNA synthesis also prevented the cell death. By contrast, nonthiol‐containing reducing agents and super oxide dismutase (SOD), an active oxygen‐eliminating enzyme, were ineffective. Thiol compounds appeared to exert a supportive level in TIG‐1 cells cultured in FBS, whereas protein synthesis inhibitors did not alter the reduced intracellular thiol content. Fragmentation of DNA occurred prior to the plasma membrane breakdown of dying cells. Taken together, these data suggest that serum‐induced cell death represents a form of apoptosis in which molecules containing thiol groups are active participants. © 1994 Wiley‐Liss, Inc.
Purification of a cell growth factor from a human lung cancer cell line: Its relationship with ferritin
Authors: Kikyo Nobuaki , Hagiwara Koichi , Fujisawa Michio , Kikyo Nobuko , Yazaki Yoshio , Okabe Tetsuro
Published: February 2005

We have purified a cell growth factor from a human lung cancer cell line, T3M‐30, which was established in a protein‐free chemically defined medium. The factor, designated carcinoma‐derived growth factor (CD‐GF), stimulated proliferation of a variety of cells, including human leukemia cells, HL‐60, and melanoma cells, SK‐28. Half‐maximum stimulation by the purified CD‐GF was achieved at a concentration of 40 ng/ml. In the purified CD‐GF, two major protein bands of 24 kDa and 22 kDa were identified on a SDS polyacrylamide gel. The partial amino acid sequences of the 24 kDa protein were determined from two peptide fragments obtained by V8 protease treatment. The partial sequences were identical to those of heavy chain of human ferritin. The activity of the purified CD‐GF was coprecipitated completely with a monoclonal antibody to heavy chain of ferritin. Ferritin has been considered to inhibit cell growth. However, human heart ferritin was capable of stimulating the growth of HL‐60 cells. These results suggest that CD‐GF is related to feritin and ferritin is a growth factor of HL‐60 leukemia cells. © 1994 Wiley‐Liss, Inc.
Interaction of high‐molecular‐weight basic fibroblast growth factor with endothelium: Biological activity and intracellular fate of human recombinant Mr 24,000 bFGF
Authors: Gualandris A. , Urbinati C. , Rusnati M. , Ziche M. , Presta M.
Published: February 2005

The single‐copy gene of human basic fibroblast growth factor (bFGF) encodes four co‐expressed isoforms, with an apparent molecular weight (Mr) of 24kD, 22.5 kD, 22kD, and 18kD, co‐translated from a single mRNA. As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24‐kD bFGF was produced and purified from transformed Escherichia coli cells. To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24‐kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS‐1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24‐kD bFGF, which localizes in the cell nucleus as an intact protein. When the same 24‐kD bFGF, cDNA was expressed in E. coli, the recombinant protein was purified to homogeneity by heparin‐Sepharose and ion‐exchange chromatography. Recombinant 24‐kD bFGF was similar to recombinant 18‐kD bFGF in receptor‐binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells. In agreement with the in vitro observations, 24‐kD bFGF and 18‐kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea. Experiments performed with the radiolabeled molecule demonstrated that 24‐kD bFGF has an intrinsic ability to bind to high‐affinity receptors when added to endothelial GM 7373 cell cultures. Receptor‐bound 24‐kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 13‐kD bFGF. Internalized 24‐kD bFGF is first processed to the 18‐kD form via a chloroquine‐insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfected COS‐1 cells, only limited amounts of exogenous internalized 24‐kD bFGF associates with the nucleus in the intact form, mostly of the nuclear‐bound molecule being represented by the processed 18‐kD protein and by smaller degradation products. In conclusion, human recombinant 24‐kD bFGF exerts a biological response in endothelial cells similar to 18‐kD bFGF both in vitro and in vivo. Our data point to a different intracellular behavior of the high‐molecular‐weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells. © 1994 Wiley‐Liss, Inc.
Proliferation and differentiation of rat adipose precursor cells in chemically defined medium: Differential action of anti‐adipogenic agents
Authors: Vassaux Georges , Négrel Raymond , Ailhaud Gérard , Gaillard Danielle
Published: February 2005

Primary rat adipose precursor cells, maintained in the minimal chemically defined medium (ITT medium) able to promote differentiation, have been used to investigate the ability of several agents to modulate their proliferation and their differentiation. Fetuin and fibroblast growth factor (FGF), which exhibited a strong and a weak mitogenic activity, respectively, do not significantly affect the proportion of differentiated cells as indicated by glycerol‐3‐phosphate dehydrogenase (GPDH) activity values. In contrast, carbaprostacyclin (cPGI2), a stable analogue of prostacyclin, behaves as a true adipogenic factor leading to a 4 to 5‐fold increase in GPDH‐specific activities with no significant effect on cell growth. Submaxillary gland kallikrein (SMGK), transforming growth factor‐β (TGF‐β) and tumor necrosis factor‐α (TNF‐α) behave as growth‐promoting agents but at the same time elicit a dose‐dependent inhibition of differentiation. Epidermal growth factor (EGF) and prostaglandin F2α (PGF2α) do not show any effect on cell proliferation at concentrations which exert a maximal inhibitory action on differentiation. Upon removal of EGF from the culture medium, complete resumption of differentiation occurs, whereas upon removal of PGF2α or SMGK, complete resumption only takes place when differentiation is triggered by cPGl2. Upon removal of TNF‐α, a partial resumption of differentiation is observed, whereas no subsequent differentiation is observed upon TGF‐β removal. These results emphasize the adipogenic, nonmitogenic role of cPGl2 and also allow the distinction between the various adipogenic/mitogenic factors which affect adipose cell differentiation. © 1994 Wiley‐Liss, Inc.
IGF‐II is more active than IGF‐I in stimulating L6A1 myogenesis: Greater mitogenic actions of IGF‐I delay differentiation
Authors: Ewton Diana Z. , Roof Suzette L. , Magri Karen A. , McWade Frank J. , Florini James R.
Published: February 2005

Mitogens are generally thought to inhibit myogenesis, and many cell biologists have found it hard to interpret observations that the insulin‐like growth factors (IGSs) stimulate bothe proliferation and differentiation of muscle cells in culture. Our previous studies suggested that the Type I IGF receptor mediates these actions. However, IGF‐II and insulin treatment caused myoblasts to differentiate much more extensively, suggesting that more complex mechanism; may be involved. Here we present evidence that the greater mitogenic activity of IGF‐l (compared to IGF‐ll and insulin) delays L6A 1 myoblast differentiation. Under conditions in which the mitogenicactions of IGDD‐l are suppressed, the stimulation of myogenesis by IGF‐I approached that by IGF‐II:; (1) in L6A1 cultures plated at a higher cell density; (2) in L6A1 cultured in which cell proliferation was inhibited by cytosine arabinoside or aphidicilin; and (3) in cultures of primary human muscle cells, which exhibit a smaller mitogenic responseee to IGF‐I. Further Further evidenceee that the Type Ireceptor plays a major role in relaying the signal for differentiation was obtained by suing IGF‐I and IGF‐II analogs. Analogs which have reduced affinity for the Type I receptor showed a dramatic decrease in activity, while an analog with increased affinity for the Type II receptor was no more active than native IGF‐I. Our results indicate that both mitogenic and myogenic actions of IGF‐I are mediated by the Type I receptor. We conclude that IGF‐I delays the onset of myogenesis as a result of its mitogenic actions, and only subsequenlty stimulates myogenesis. These observations reconcile the apparent conflict between our results with the IGFs and other investigator' reports of effects of other mitogens. © 1994 Wiley‐Liss, Inc.
Expression and phosphorylation of NHE1 in wild‐type and transformed human and rodent fibroblasts
Authors: McSwine Rebecca L. , Babnigg Gyorgy , Musch Mark W. , Chang Eugene B. , Villereal Mitchel L.
Published: February 2005

Activation and phosphorylation of the Na+/H+ exchanger occurs with a diverse group of mitogens such as phorbol myristate acetate (PMA), epidermal growth factor (EGF), thrombin and serum (Sardet et al., 1990, Science, 247:723‐‐726). Some of these growth factors have been shown to differentially activate the Na+/H+ exchanger in various fibroblasts (Etscheid et al., 1990, Am. J. Physiol., 259:C549‐‐C556; Muldoon, L. L., et al., 1987, Am. J. Physiol., 253:C219‐‐C229). However, alterations in the expression and phosphorylation of NHE1 in various fibroblasts has not been examined with respect to a potential mechanism of differential activation of the exchanger. To pursue this question, a novel antibody, anti‐XB17, directed to the cytoplasmic tail of NHE1 was characterized and then utilized to examine the expression of NHE1 protein and the level of phosphorylation of the serum stimulated exchanger in human embryonic lung fibroblasts (WI‐38), SV40‐transformed WI‐38, and nontransformed human foreskin fibroblasts (HSWP) cells. The level of mRNA expressed in these cells was also examined. Results indicate that the parental cell lines and other nontransformed fibroblasts express NHE1. Although the transformed cell lines express NHE1 mRNA in approximately similar abundance to the parental lines, they contain decreased quantity of NHE1 exchanger/mg membrane protein as recognized by anti‐XB17 Ab. The mechanism that results in the apparent decrease in NHE1 protein levels in the transformed cells is not known. Also, the SV40‐transformed cells express and exchanger with a higher apparent molecular weight. The WI‐38 cells demonstrate phosphorylation of NHE1 in response to mitogenic stimulation. Although the nontransformed HSWP cells have a high level of Na+/H+ exchanger protein, they do not show a significant increase in phosphorylation following serum stimulation, when examined by immunoprecipitation, and analysis on 1‐D gels. However, subsequent studies of tryptic phosphopeptides from the immunoprecipitated exchanger reveal that serum‐stimulated phosphorylation of one tryptic peptide does occur but may be masked in the first dimension by differential phosphorylation of other tryptic peptides that are more heavily phosphorylated in unstimulated cells. © 1994 Wiley‐Liss, Inc.
Bombesin stimulates the in vitro growth of a human gastric cancer cell line
Authors: Bold Richard J. , Lowry Patrick S. , Ishizuka Jin , Battey James F. , Townsend Courtney M. , Thompson James C.
Published: February 2005

Bombesin (BBS) and its mammalian equivalent, gastrin‐releasing peptide (GRP) exhibit diverse biological functions, including that of a neurotransmatter, a regulator of gastrointestinal hormone release, and a trophic factor for various normal and neoplastic tissues. Bombesin stimulates the growth of normal cells of the stomach, pancreas, and bronchial epithelium as well as cells in breast cancer, gastrinoma, and small cell lung cancer. The purpose of this study was to determine whether BBS regulates the growth of a human gastric cancer cell line (SIIA) in vitro, and if so, to examine the mechanisms of signal‐transduction that are involved. We found that BBS stimulated the growth of SIIA cells in vitro. The GRP receptor antagonists, BIM 26189 and BIM 26226, had no effect on growth of SIIA cells. Although these antagonists blocked the BBS‐induced increase of [Ca2+]i, they failed to block the growth‐stimulatory effect of BBS. BBS stimulated intracellular tyrosine phosphorylation of multiple proteins, with a predominant protein of apparent molecular weight of 125 kDa. Inhibition of intracellular tyrosine kinases by tyrphostin blocked the growth‐stimulatory effect of BBS on SIIA cells. These results indicate that BBS exerts its trophic effect on SIIA cells through a receptor(s) linked to tyrosine kinase pathway, but not to the phospholipase C (PLC) pathway. © 1994 Wiley‐Liss, Inc.
Selective effects of the PKC inhibitors Ro 31‐8220 and CGP 41 251 on PMN locomotion, cell polarity, and pinocytosis
Authors: Keller H. U. , Niggli V.
Published: February 2005

Using two newly synthesized inhibitors, Ro 31‐8220 and CGP 41 251, of protein kinase C (PKC), we analyzed: (1) how distinct PMN functions (shape changes, locomotion, pinocytosis) are regulated, and (2) the role of protein phosphorylation and PKC in this process. We were able to transform: (1) resting PMNs into locomoting cells using fNLPNTL, (2) locomoting cells into non‐locomoting highly pinocytic cells using PMA, and (3) PMA‐stimulated cells showing marked pinocytosis into locomoting or into resting cells using Ro 31‐8220. It is thus possible to selectively manipulate PMN function (resting state, locomotion, marked pinocytosis), indicating that there are different regulatory pathways. It was not possible to induce locomotion and marked pinocytosis simultaneously, indicating crosstalk between pathways. Ro 31‐8220 inhibited PMA‐induced shape changes (nonpolar cells) and pinocytosis, but not fNLPNTL‐induced shape changes (polarity) and pinocytosis. At higher concentrations, Ro 31‐8220 alone elicited cell polarity and chemokinesis, indicating that a constitutively active protein kinase is involved in maintaining the spherical shape of resting PMNs. Functional effects of another PKC inhibitor, CGP 41 251, on neutrophil function were strikingly different. CGP 41 251 selectively inhibited fNLPNTL‐induced polarity and locomotion (but not colchicine or Ro 31‐8220‐induced polarity), and it failed to inhibit PMA‐induced, stimulated pinocytosis and shape changes. Although the effects of Ro 31‐8220 vs. CGP 41 251 on PMN function were strikingly different, the inhibition of profiles for constitutive and for fNLPNTL‐ or PMA‐induced protein phosphorylation in intact PMNs showed only small differences, which could not yet be conclusively related to cell function. © 1994 Wiley‐Liss, Inc.
Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide
Authors: Betz Natalie A. , Fattaey Heideh K. , Johnson Terry C.
Published: February 2005

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS‐18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixtyfold greater sensitivity than other fibroblasts and epithelial‐like cells to CeReS‐18‐induced growth inhibition. Growth inhibition induced by CeReS‐18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS‐18‐induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS‐18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS‐18‐induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS‐18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS‐18‐induced growth arrest. The CeReS‐18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling. © 1994 Wiley‐Liss, Inc.
Activation of TNF‐R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells
Authors: Wada H. Garrett , Fok Katherine S. , Fendly Brian M. , Chiang Nancy Y. , Sussman Howard H.
Published: February 2005

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF‐α has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM‐SS cells). CEM‐SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2‐4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o‐phenanthroline markedly potentiated the effects of TNF on the resistant CEM‐SS cells leading to cell death. This observation was specific for copper because ferric o‐phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF‐R1 receptor and independent of phospholipase A2 signaling. © 1994 Wiley‐Liss, Inc.
Immunolocalization of mitochondrial 3‐hydroxy‐3‐methylgutaryl CoA synthase in rat liver
Authors: Royo Teresa , Pedragosa Maria J. , Ayté José , Gil‐Gómez Gabriel , Vilaró Senén , Hegardt Fausto G.
Published: February 2005

We report the preparation of specific polyclonal antibodies raised against two synthetic peptides deduced from the cDNA sequence for the rat liver mitochondrial 3‐hydroxy‐3‐methylglutaryl Coenzyme A (HMG‐CoA) synthase gene. Immunoelectron microscopy using these antibodies on hepatic cryoultrathin sections confirms the mitochondrial localization of this protein in hepatocytes. Immunofluorescence microscopy on frozen sections of adult rat liver revealed fluorescence inside all hepatocytes, with no evidence of zonation, indicating that ketogenesis may not be limited to specific regions of rat liver but is extended to all hepatocytes. © 1995 Wiley‐Liss, Inc.
Tumor necrosis factor reduces proteoglycan synthesis in cultured endothelial cells
Authors: Ramasamy S. , Lipke D. W. , McClain C. J. , Hennig B.
Published: February 2005

Tumor necrosis factor (TNF)‐induced disruption of vascular endothelial barrier function may be due in part to alterations in proteoglycan metabolism. To test this hypothesis, confluent endothelial cell monolayers were exposed for 24 h to 500 or 1,000 U of TNF per mililiter of culture medium together with 20 μCi Na2 35SO4. HPLC anion‐exchange separation of proteoglycans secreted into media of control as well as TNF‐treated cultures revealed one major peak (representing 95% of total radioactivity) and one minor peak (representing 5% of total radioactivity), which eluted at 0.6 and 0.9 M NaCl, respectively. One single peak was obtained from control as well as TNF‐treated endothelial cell monolayers and eluted at 1.2 M NaCl. TNF treatment did not change the total quantity of radioactive proteoglycans secreted into the media but significantly decreased the amount of proteoglycans in endothelial cell monolayers. However, TNF treatment did not alter the size or glycosaminoglycan (GAG) composition of the proteoglycans either in the media or in the cell monolayers. In addition, mRNA levels of specific proteoglycans, perlecan and biglycan, were measured upon TNF treatment, using Northern analysis. TNF treatment caused a dose‐dependent decrease in mRNA levels for the biglycan in endothelial cultures. These results suggest that TNF decreases production of proteoglycans and alters normal endothelial cell proteoglycan metabolism which may be sufficient to impair endothelial barrier function. © 1995 Wiley‐Liss, Inc.
New CD44 splice variants associated with human breast cancers
Authors: Iida Naoko , Bourguignon Lilly Y. W.
Published: February 2005

Changes in the CD44 variant (CD44v) isoforms on the cell surface have been correlated with tumor metastasis. In this study we have examined the expression of CD44 variant isoforms in human breast carcinoma samples by a variety of techniques including immunohistochemistry, reverse transcriptase‐polymerase chain reaction (RT‐PCR), and nucleotide sequencing. Using RT‐PCR, we have determined that normal human breast tissue contains primarily the CD44 epithelial (CD44E) form and very little CD44 standard (CD44s) form. However, metastatic breast carcinomas appear to overxpress both the CD44E and CD44s forms and also display multiple new species of CD44 variant isoforms. Histocytochemical staining using anti‐CD44 antibody (recognizing a common determinant of the CD44 class of glycoproteins) confirms that the CD44 molecules are overexpressed and preferentially located in metastatic breast cancer tissues. Nucleotide sequencing analyses indicate that at least four new CD44 variant isoforms (i. e., displaying unique splicing via the insertion or the deletion of exons 7, 10, 11, and 14) may be closely associated with human metastatic breast cancers. These newly described CD44 variant isoforms may be useful for monitoring the progression of human breast cancer metastasis. © 1995 Wiley‐Liss, Inc.
Multiple sites of vanadate and peroxovanadate action in Xenopus oocytes
Authors: Barnes David M. , Sykes Destiny B. , Shechter Yoram , Miller David S.
Published: February 2005

In Xenopus laevis oocytes, the insulin mimics, vanadate and peroxovandates (PV), stimulated the uptake of 3H‐2‐deoxyglucose and incorporation of 35S‐methionine into protein. For both hexose transport and protein synthesis, peroxovandates (produced by reacting vandate and H2O2) were at least as potent as vandate. Microinjection of peroxovandates into the oocytes stimulated 2‐deoxyglucose uptake. However, methionine incorporation was not stimulated by microinjection of peroxovanadate or vanadate solutions. Consistent with these results and with the possibility that vandate and peroxovandates enter the cell on a phosphate transporter, raising the medium phosphate concentration from 1 mM to 10 mM blocked vanadate‐stimulated hexose transport and partially reduced peroxovanadates stimulation of hexose transport. Increased medium phosphate did not reduce stimulation of protein synthesis by either effector. Taken together, these data indicate that vanadate/peroxovanadates act at both intracellular and extracellular sites. Action at the former stimulates hexose uptake and action at the latter, protein synthesis. © 1995 Wiley‐Liss, Inc.

This artilce is a US Government work and, as such, is in the public domain in the United States of America.
1,25‐dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: An effect modulated by vitamin D status
Authors: Wali Ramesh K. , Bissonnette Marc , Jiang Kenneth , Niedziela Sharon M. , Khare Sharad , Roy Hemant K. , Sitrin Michael D. , Brasitus Thomas A.
Published: February 2005

Our laboratory has recently demonstrated that 1,25‐dihydroxyvitamin D3(1,25(OH)2D3) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D‐sufficient rat colonocytes. These effects of 1,25(OH)2D3 were, however, lost in vitamin D‐insufficient rats and restored by the in vivo repletion of 1,25(OH)2D3. In the present studies we have examined the ability of 1,25(OH)2D3 to stimulate the phosphorylation of colonic membrane proteins in intact D‐sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)2D3 increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D‐sufficient rats. Moreover, in vitamin D‐sufficient rats, treatment of colonocytes with 1,25(OH)2D3 or 12‐Otertradecanoyl phorbol 13‐acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)2D3 were both rapid and transient. In addition, PKC19–36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)2D3. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D‐sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D‐insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)2D3‐treatment of colonocytes from vitamin D‐insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)2D3 following the in vivo repletion of vitamin D‐insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)2D3 treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. © 1995 Wiley‐Liss, Inc.
Iron transport mechanisms in reticulocytes and mature erythrocytes
Authors: Hodgson Leah L. , Quail Elizabeth A. , Morgan Evan H.
Published: February 2005

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with 59Fe‐labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration‐dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin‐bound Fe(II) by two mechanisms. One is a high‐affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low‐affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly assoicated with exchange of Na+. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)‐sucrose and Fe(II)‐ascorbate using a variety of incubation conditions. No major differences were observed. © 1995 Wiley‐Liss, Inc.
Down‐regulation of bradykinin receptors and bradykinin‐induced Ca2+ mobilization, tyrosine phosphorylation, and DNA synthesis by autocrine factors, tumor necrosis factor α, and interferon β in Swiss 3T3 cells
Authors: Kiehne Karlheinz , Rozengurt Enrique
Published: February 2005

Preincubation of quiescent Swiss 3T3 cells in fresh synthetic medium caused a reduction of the lag period prior to bradykinin‐stimulated DNA synthesis as well as a leftward shift in the dose‐response curve (half‐maximum effect at 2 nM and 8 nM for preincubated cells and control cells, respectively). These enhancing effects were selective for bradykinin since vasopressin‐stimulated DNA synthesis was not affected by preincubation in synthetic medium. Preincubation in synthetic medium also caused a marked enhancement (five‐ to sixfold increase) of bradykinin‐induced Ca2+ mobilization from intracellular stores. This enhancement was time‐dependent, peaked after 12 h of preincubation, and was prevented by inhibition of RNA or protein synthesis. Furthermore, preincubation in synthetic medium did not enhance the Ca2+ mobilization by bombesin, vasopressin, or PDGF. Additionally, bradykinin‐induced tyrosine phosphorylation was also enhanced by prior incubation in fresh medium. Scatchard analysis of [3H]bradykinin binding revealed a doubling of the number of bradykinin receptors without any significant change of affinity after preincubation, thus providing an explanation for the increased cellular responsiveness to bradykinin. This enhancement of responsiveness to bradykinin was caused by the removal of an inhibitory factor present in conditioned medium which is produced by the cells and accumulates gradually in the medium. Addition of tumor necrosis factor α or interferon β to synthetic medium substituted for conditioned medium in preventing the increase in responsiveness to bradykinin. These findings demonstrate a novel mechanism that regulates cellular sensitivity to bradykinin via an autocrine factor(s). © 1995 Wiley‐Liss, Inc.
In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells
Authors: Popnikolov Nikolay K. , Yang Jason , Guzman Raphael C. , Swanson Steven M. , Thordarson Gudmundur , Collins Gail , Talamantes Frank , Nandi Satyabrata
Published: February 2005

A new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo‐2′‐deoxy‐uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17β‐estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice. © 1995 Wiley‐Liss, Inc.
Mechanical loading stimulates the release of transforming growth factor‐β activity by cultured mouse calvariae and periosteal cells
Authors: Klein‐Nulend Jenneke , Roelofsen Jan , Sterck Jozien G. H. , Semeins Cornelis M. , Burger Elisabeth H.
Published: February 2005

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) inhibits bone resorption and stimulates bone formation in cultured fetal mouse calvariae (Klein‐Nulend et al., 1986, Arthritis Rheum., 29:1002–1009). The production of soluble bone factors by such calvariae is also modified (Klein‐Nulend et al., 1993, Cell Tissue Res., 271:513–517). Transforming growth factor‐β (TGF‐β) is an important local regulator of bone metabolism and is produced by osteoblasts. In this study, the release of TGF‐β activity as a result of mechanical stress was examined in organ cultures of neonatal mouse calvariae, in primary cultures of calvariae‐derived osteoprogenitor (OPR) cells, and in more differentiated osteoblastic (OB) cells. Whole calvariae and calvariaederived cells were cultured in the presence or absence of IHC for 1–7 days and medium concentrations of active as well as total TGF‐β were measured using a bioassay. IHC (maximum 13 kPa, maximal pressure rate 32.5 kPa/sec) was generated by intermittently (0.3 Hz) compressing the gas phase above the cultures. We found that mechanical loading by IHC stimulated the release of TGF‐β activity from cultured calvariae by twofold after 1 day. IHC also stimulated the release of TGF‐β activity from calvariae‐derived cells after 1 and 3 days. The absolute amounts of TGF‐β activity released were lower in OPR cells than in OB cells, but the stimulatory effect of IHC was greater in OPR cells. Total TGF‐β (active and bound) released into the medium was not affected by IHC. IHC did not change the dry weight of the organ cultures, nor the DNA or protein content of the cell cultures. These data show that mechanical perturbation of bone cells, particularly OPR cells, enhances the activation of released TGF‐β. We conclude that modulation of TGF‐β metabolism may be part of the response of bone tissue to mechanical stress. © 1995 Wiley‐Liss, Inc.
Okadaic acid, cAMP, and selected nutrients inhibit hepatocyte proliferation at different stages in G1: Modulation of the cAMP effect by phosphatase inhibitors and nutrients
Authors: Mellgren Gunnar , Vintermyr Olav K. , Døskeland Stein O.
Published: February 2005

The protein phosphatase inhibitor okadaic acid (> 100 nM) caused an abrupt and complete cessation of primary rat hepatocyte cell cycle progression at the restriction point in late G1. A decline in the G1/S transition rate was observed in response to elevated cAMP, excess selected nutrients, and okadaic acid (< 100 nM). Excess nutrients (40 mM glucose ± 5 mM dihydroxyacetone) acted by imposing an incomplete block in early G1. The cAMP action was potentiated by the phosphatase inhibitor microcystin, which in itself did not affect DNA replication. This suggests that cAMP acted by phosphorylating substrate(s) that is dephosphorylated by a microcystin‐sensitive phosphatase. The additive effects of submaximal concentrations of okadaic acid and cAMP analogs indicated that okadaic acid and cAMP acted via different pathways. In conclusion, okadaic acid, cAMP, and excess nutrients, acting through distinct pathways, inhibited hepatocytes in different parts of the G1 phase. © 1995 Wiley‐Liss, Inc.