bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective increase of c‐myc mRNA levels by methylglyoxal‐bis (guanylhydrazone) and novobiocin in serum‐stimulated fibroblasts
Authors: Aller Patricio , Baserga Renato
Published: February 2005

We have studied the effect of methylglyoxal‐bis (guanylhydrazone) (MGBG) and novobiocin on the accumulation of specific mRNAs in serum‐stimulated ts13 cells (a temperature‐sensitive mutant of the BHK cell line). The RNAs studied included: c‐myc, v‐ras, ornithine decarboxylase, β‐actin, histone H3, and those represented by clones p2F1 and p1B6 (Hirschhorn et al., Proc. Natl, Acad. Sci. USA, 81: 6004, 1984) All these RNAs accumulated at higher levels when quiescent cells were serum stimulated for 16 h. Both MGBG (25 μM and 100 μM) and novobiocin (200 μg/ml) effectively prevented the transition from G0 to S phase. We found that 100 μM MGBG induced an overaccumulation of c‐myc RNA while H3 RNA was decreased, and the steady‐state levels of all other RNAs were the same as in cells stimulated without the drug. Novobiocin prevented the serum‐induced increase in the amount of all RNAs, which remained at the same levels as in quiescent cells, with the exception of c‐myc, which again accumulated at a higher level in drug‐treated cells than in serum‐stimulated untreated cells. The possible significance of these results is discussed.
Temperature ranges over which rainbow trout fibroblasts survive and synthesize heat‐shock proteins
Authors: Mosser D. D. , Heikkila J. J. , Bols N. C.
Published: February 2005

Cultures of the rainbow trout fibroblast cell line RTG‐2 withstood temperatures from 0°C to 28°C. At 0°C and 28°C, no prolifertion occurred, but cells persisted for at least 7 days. If the cultures were placed back at 22°C, proliferation returned to normal in those that had been kept at 0°C but was reduced in cultures that had been kept at 28°C. Above 28°C, cultures survived for only short periods. If RTG‐2 cells that were grown routinely at 22°C were shifted to 26, 28, and 30°C, heat shock proteins (hsps) of 100, 87, 70, 68, 60, 39, 27, and 19 kilodaltons were synthesized. Synthesis was most pronounced at 28°C, and at this temperature hsp synthesis was maximal by 2 hr and had returned to control levels by 36 hr. Individual hsps were synthesized maximally at slightly different times and temperatures, but under all conditions hsps 87 and 70 were most abundant. If cultures were shifted to 24°C or 32°C, hsp synthesis was not observed. Neither the placement of cultures at 5°C nor the shift of cultures that had been maintained at 0°C or 5°C back to 22°C induced the synthesis of hsps. However, cultures incubated at 5°C for 24 hr did synthesize hsps at 26°C, 28°C, and 30°C.
Intracellular location of the Ah receptor
Authors: Gudas Jean M. , Karenlampi Sirpa O. , Hankinson Oliver
Published: February 2005

The subcellular distribution of the Ah receptor from the mouse hepatoma line, Hepa‐1, was investigated following cytochalasin B treatment and cell enucleation. Probing the resultant cytoplast and nucleoplast fractions with radiolabelled tetrachlorodibenzo‐p‐dioxin (TCDD) revealed the presence of a specifically bound peak of receptor only in the cytoplast fraction. However, the quantity of receptor recovered in these experiments was only 10–12% of the expected value. We therefore undertook an investigation to determine the fate of the Ah receptor in the presence of cytochalasin B. Incubation of Hepa‐1 cells with this compound resulted in a rapid loss or inactivation of cytosolic binding activity with a concomitant decrease in the amount of receptor partitioned into the nucleus at all time periods examined. Control experiments indicated that cytochalasin B did not compete with TCDD for binding to the Ah receptor and furthermore, that its mechanism of action could not be attributed to a non‐specific effect on all cytosolic proteins. The results obtained are discussed in relation to the proposed models for induction by the estrogen and glucocorticoid binding receptors.
Persistent infection with a nontransforming RNA virus leads to impaired growth factor receptors and response
Authors: Verdin Eric M. , Maratos‐Flier Eleftheria , Carpentier Jean‐Louis , Kahn C. Ronald
Published: February 2005

The potential role of viral persistence with nontransforming viruses on cellular growth and cellular function has received little attention. We found that when infected with type 3 reovirus (five plaque‐forming units (PFU)/cell), balb/C 3T3 cells (a mouse embryo fibroblast cell line) undergo a limited lytic phase. The surviving cells, about 90% of the original cells, appear morphologically normal by light microscopy and exhibit normal growth patterns in serum‐supplemented medium but are persistently infected by electron microscopy. These persistently infected cells shed infectious virus in the culture medium (1.6–60 × 106 PFU per 106 cells per 24 h). In comparison to control uninfected 3T3 cells, the persistently infected cells exhibit a 70–90% decrease in receptor number for epidermal growth factor (EGF). This occurs without production of any EGF‐like material and is associated with a parallel decrease in EGF‐stimulated DNA synthesis. By contrast, insulin receptors are increased in number three‐fold and insulin and serum stimulated DNA synthesis are comparable to control uninfected cells. These results suggest that persistent infection with a nontransforming virus may lead to major alteration in control of cell growth by specific growth factors.
Sodium 22+ washout from cultured rat cells
Authors: Kino Minoru , Nakamura Akitoshi , Hopp Laszlo , Kuriyama Satoru , Aviv Abraham
Published: February 2005

The washout of Na+ isotopes from tissues and cells is quite complex and not well defined. To further gain insight into this process, we have studied 22Na+ washout from cultured Wistar rat skin fibroblasts and vascular smooth muscle cells (VSMCs). In these preparations, 22Na+ washout is described by a general three‐exponential function. The exponential factor of the fastest component (k1) and the initial exchange rate constant (kie) of cultured fibroblasts decrease in magnitude in response to incubation in K+ ‐deficient medium or in the presence of ouabain and increase in magnitude when the cells are incubated in a Ca++ ‐deficient medium. As the magnitude of the kie declines (in the presence of ouabain) to the level of the exponential factor of the middle component (k2), 22Na+ washout is adequately described by a twoexponential function. When the kie is further diminished (in the presence of both ouabain and phloretin) to the range of the exponential factor of the slowest component (k3), the washout of 22Na+ is apparently monoexponential. Calculations of the cellular Na+ concentrations, based on the 22Na+ activity in the cells at the initiation of the washout experiments, and the medium specific activity agree with atomic absorption spectrometry measurements of the cellular concentration of this ion. Thus, all three components of 22Na+ washout from cultured rat cells are of cellular origin. Using the exponential parameters, compartmental analyses of two models (in parallel and in series) with three cellular Na+ pools were performed. The results indicate that, independent of the model chosen, the relative size of the largest Na+ pool is 92–93% in fibroblasts and approximately 96% in VSMCs. This pool is most likely to represent the cytosol.
Heparin inhibition of smooth muscle cell proliferation: A cellular site of action
Authors: Reilly Christopher F. , Fritze Linda M. S. , Rosenberg Robert D.
Published: February 2005

The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet‐derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin‐Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non‐PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.
Activated N‐ras gene induces neuronal differentiation of PC12 rat pheochromocytoma cells
Authors: Guerrero Isabel , Wong Henry , Pellicer Angel , Burstein David E.
Published: February 2005

Activated mouse N‐ras gene transfected into PC12 rat pheochromocytoma cells suppressed proliferation and promoted neuronal differentiation. Normal mouse N‐ras in a LTR‐containing vector caused differentiation with a reduced efficiency, but normal N‐ras in a vector lacking LTR sequences failed to alter the PC12 phenotype. Cultures of NGF‐resistant PC12 variant subline U7 also showed outgrowth of neurites and cessation of cell division following transfection with the mutated ras gene. The present findings suggest that ras genes can, in certain cells, play a role in promoting differentiation and suppressing proliferation, in contrast to their established oncogenic neoplasiapromoting activity in other cells.
Dexamethasone and dimethylsulfoxide as distinct regulators of growth and differentiation of cultured suckling rat hepatocytes
Authors: Baribault Hélène , Marceau Normand
Published: February 2005

Dexamethasone can promote the differentiation of different tissues in vivo while dimethylsulfoxide is a commonly used inducer of differentiation in various tumor cell types in culture. In the present study, the effects of dexamethasone and dimethylsulfoxide on growth and functional activities of cultured differentiating suckling rat hepatocytes stimulated with various combinations of EGF, insulin, and glucagon were evaluated. Hepatocytes stimulated with ECF and either insulin or glucagon entered S phase and mitosis after a lag period of 24 h. These hormonal factors thus provide simple combinations of hepatocyte‐growth regulators. Dexamethasone in the presence of EGF and glucagon inhibited the initiation of DNA synthesis and mitosis, but it had no effect on EGF‐insulin stimulated cultures. Such a differential effect of dexamethasone was observed at concentrations ranging from 4 nM to 200 μM. α‐Fetoprotein, albumin, and tyrosine aminotransferase were used as typical markers of hepatocyte differentiation status. Irrespective of the combinations of growth‐promoting factors used, dexamethasone inhibited α1‐fetoprotein production and maintained albumin production and tyrosine aminotransferase inducibility. In contrast, dimethylsulfoxide at 2% inhibited hepatocyte growth and supported the maintenance of the production of both α1‐fetoprotein and albumin, independent of the hormonal growth regulators used. On this basis, dexamethasone and dimethylsulfoxide act as distinct modulators of growth and maturation of cultured differentiating suckling rat hepatocytes.
Changes of serum‐induced ornithine decarboxylase activity and putrescine content during aging of IMR‐90 human diploid fibroblasts
Authors: Chenc Kuang Yu , Chang Zee‐Fen , Liu Alice Y.‐C.
Published: February 2005

The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum‐induced changes of these parameters in quiescent IMR‐90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR‐90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum‐induced ODC activity and DNA synthesis in IMR‐90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNa synthesis in young cells (PDL = ∼ 18–22) were, respectively, five‐fold and six‐fold greater than that in old cells (PDL = ∼ 50–55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR‐90 cells did not show significant PDL‐dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR‐90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR‐90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR‐90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR‐90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum‐induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson‐Cilford syndrome.
Stimulation of bovine endothelial cell angiotensin‐I‐converting enzyme activity by cyclic AMP‐related agents
Authors: Krulewitz A. H. , Fanburg B. L.
Published: February 2005

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (β adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin‐I‐converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10−3 M) but not cAMP or dibutyryl cGMP stimulated angiotensin‐l‐converting enzyme (ACE) activity of cells in culture approximately 50–100% but had little influence on ACE activity of the medium. Theophylline at 10−3 M concentration produced a three‐ to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10−7‐10−5 M concentration after pretreatment of cells for 24 hr with 10−4 M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls.
Effect of inositol hexaphosphate on the transient behavior of red cells following a DMSO‐induced osmotic pulse
Authors: Franco Robert S. , Barker Robin , Novick Steven , Weiner Murray , Martelo Orlando J.
Published: February 2005

An osmotic pulse can be used to incorporate inositol hexaphosphate (IHP) into red cells. The pulse is induced by equilibrating a red cell suspension with DMSO and then rapidly diluting with an isotonic IHP solution. Since IHP binds to hemoglobin and lowers the affinity for oxygen, this method may find application in the preparation of low‐affinity cells for experimental and clinical use. The experiments reported here examined the dynamic changes of several red cell variables immediately following the osmotic pulse. The effect of IHP, which has been shown to dissociate red cell cytoskeletons, was evaluated by comparison with a matched phosphate‐buffered saline (PBS) diluent. Red cell morphology, volume, and hemoglobin permeability were studied by fixing the cells at times ranging from 0.06 to 300 sec after dilution. Mechanical fragility was measured by subjecting the cells to a short period of shear stress at the same times after dilution. With both diluents, the cells underwent a rapid increase in volume followed by a return towards normal volume with a maximum at less than 250 msec. With IHP diluent, the period of hemoglobin permeability immediately followed the size peak and was completed by about 1 sec after dilution. PBS also induced a second leakage at longer times (10–120 sec), which resulted in a morphological dichotomy with ghosts and intact cells. The choice of diluent also affected sensitivity to shear stress. The IHP‐treated cells had a mechanical fragility maximum at about 1 sec. The PBS‐treated cells exhibited no enhanced mechanical fragility. An unexpected result was the inhibition of the second phase of lysis in PBS‐treated cells by a properly timed shear stress.
Osteoclasts and osteoblasts migrate in opposite directions in response to a constant electrical field
Authors: Ferrier Jack , Ross Stephen M. , Kanehisa Junya , Aubin Jane E.
Published: February 2005

We have investigated in vitro the effects of the electrical field produced by constant current on freshly isolated rabbit osteoclasts and on well characterized clonal rat osteoblastlike cells. At field strengths of 0.1 and 1 V/mm, the osteoclasts migrated rapidly toward the positive electrode, whereas the osteoblastlike cells migrated in the opposite direction, toward the negative electrode. Thus, different cell types from the same tissue can respond differently to the same electrical signal. These results have important implications for hypotheses concerning the cellular mechanism of galvanotaxis, and may also clarify the cellular basis of the clinical application of electrical stimulation of bone healing.
Inhibition by phorbol esters of antiimmunoglobulin induced calcium signalling and B‐cell activation
Authors: Rothstein Thomas L. , Kolber Donna L. , Simons Elizabeth R. , Baeker Thomas R.
Published: February 2005

The inhibitory effect of phorbol dibutyrate (PDB) on B‐cell stimulation was evaluated using a model in which activation is induced by modest doses of antiimmunoglobulin antibody (anti‐lg) and progression to DNA synthesis is induced by cytochalasin. PDB preferentially inhibited anti‐lg‐induced activation and did so during brief (2 hr) preincubation with anti‐lg. Activation was inhibited whether PDB was added before or shortly after anti‐lg. Since activation for cytochalasin responsiveness appears to be mediated by Ca2+, the effect of PDB on the anti‐lg‐induced rise in intracellular Ca2+ was evaluated. PDB (and other phorbol esters that activate protein kinase C) inhibited the rise in Ca2+ normally associated with anti‐lg treatment; moreover, PDB reversed an established anti‐lg‐induced Ca2+ response. These data suggest that phorbol esters inhibit B‐cell activation by interfering with the elevated levels of intracellular Ca2+ produced by cross‐linking of surface immunoglobulin by anti‐lg. This could represent a “feedback inhibition” type of response, but it remains to be seen if this occurs under physiological conditions of protein kinase C activation.
Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells
Authors: Hyslop Paul A. , Hinshaw Daniel B. , Schraufstätter Ingrid U. , Sklar Larry A. , Spragg Roger G. , Cochrane Charles G.
Published: February 2005

The effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator “Quin 2.” [Ca++]i rose from approximately 150 nM to >2 μM over a time course that was strongly dependent on the concentration of H2O2 used (5 × 10−5 to 5 × 10−3M). After exposure of P388D1 cells to 5 × 10−3 M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spetcroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points >45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 × 10−4 M H2O2 to 14.0 min at 5.0 × 10−3 M. When the total [Ca++]i‐buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68‐fold (1.1 × 102 ‐ 7.6 × 103 amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6‐fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca+++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that “surface blebs” appeared during this phase of injury. Both the rise in [Ca+++]i and “blebbing” were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, (1) H2O2 exposure induces a dose‐dependent disturbance of intracellular calcium homeostatis; (2) the rise in[Ca+++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; (3) the rate of rise of [Ca+++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; (4) during the early phase (<30 min) of rise of [Ca+++]i, only intracellular calcium is involved in the response; (5) these events occur concomitantly with gross morphological changes to the plasma membrane; and finally, (6) these events precede loss of integrity of the plasma membrane as a permeability barrier.
L‐929 cells under hyperosmotic conditions: Volume changes
Authors: Clegg James S.
Published: February 2005

Volume changes resulting from the addition of sorbitol to the environment of mouse L‐929 cells were evaluated from cell diameter measurements. Over periods of 1 hour or less, this solute was effectively excluded from intracellular water. The reduction in cell volume was inversely related to sorbitol concentration up to levels of about 0.6 molal, above which no further significant reduction occurred. Reduced cell volumes were maintained for at least 1 hour without measurable volume regulation. The percentage of volume lost was independent of the initial cell volume and was quickly regained when physiological conditions were restored. However, cell volume was influenced strongly by cell density or by some variable related to it. L‐cells store surface area when dehydrated, apparently by means of plasma membrane convolutions and microvilli, based on the rapid kinetics of reversible volume changes and on observations from scanning electron microscopy. These results are related to current views on the nature of intracellular organization.
Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: Relationship to senescence
Authors: Hornsby Peter J. , Aldern Kathy A. , Harris Sandra E.
Published: February 2005

During cellular senescence, non‐clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin‐ or prostaglandin E1− stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH‐stimulated cAMP production rates were measured in clones immediately after isolation, during long‐term growth following isolation, and after subcloning. ACTH‐stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/106 cells/min, whereas mass cultures at this PDL showed ∼50 pmol/106 cells/min. Forskolin‐stimulated cAMP production rates in clones varied only over the range of 59–119 pmol/106 cells/min and showed no correlation with the ACTH‐stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP‐inducible 17α‐hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub‐subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eightfold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.
Intracellular distribution of active and inactive transglutaminase in stimulated cultured C6 glioma cells
Authors: Korner G. , Bachrach U.
Published: February 2005

The cellular distribution of active and inactive transglutaminase (TGase) was studied in C6 glioma cells before and during stimulation by a serum‐containing medium. The activity of the enzyme was determined in the soluble and insoluble fractions obtained by freezing and thawing the cells, followed by centrifugation at 12,000g for 5 min. In the soluble fractions, the activity of TGase decreased 2.5 h post‐stimulation and increased after 5 and 8 h. In the corresponding insoluble fractions, no significant changes in the activity of the enzyme were noted up to 8 h after stimulating the cells with fresh medium. An immunological approach was next used to determine the quantity of TGase antigen during the stimulation of the cultured glioma cells. In the soluble fraction, the quantity of the antigen decreases significantly at 2.5, 5, and 8 h. In contrast, in the insoluble fraction, a significant increase in TGase antigen was detercted 8 h after the addition of fresh medium. Cycloheximide completely inhibited the increase in the quantity of TGase antigen in the insoluble fraction, 8 h post‐stimulation, while actinomycin D caused a partial inhibition. Trypsin, neuraminidase, or Sendai viruses increased the activity of TGase significantly, when added to nonstimulated cells. Trypsin had no effect on TGase activity when added to the cells 2 h after stimulation with a serumcontaining medium. These findings suggest that an inactive form of the enzyme is present in the insoluble cellular fraction. A model has been proposed to explain the variations in TGase activity, its distribution and translocation during cellular stimulation.
Murine macrophage heparanase: Inhibition and comparison with metastatic tumor cells
Authors: Savion Naphtali , Disatnik Marie‐Helene , Nevo Zvi
Published: February 2005

Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16‐BL6 melanoma; ESb T‐lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monolyaer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = ‐labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intacellularly but it is instead found pericellularly and requires a continuous cell‐matrix contract at the optimal pH for maintaining cell growth.

The degradation of [35S]O4=‐labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 μg/ml), arteparon (10 μg/ml), and heparin at a concentration of 3 μg/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo‐β‐glucuronidase D‐saccharic acid 1,4‐lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two‐step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases—α2‐macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)—failed to inhibit this degradation process, and only α1‐antitrypsin inhibited the heparanase activity.

B16‐BL6 metastatic melanoma cell heparanase, which is also a cell‐associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene‐induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 μ/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood‐borne tumor metastasis.
Regulation of amino acid transport in isolated rat hepatocytes during development
Authors: Leoni S. , Spagnuolo S. , Dini L. , Devirgiliis L. Conti
Published: February 2005

The effect of amino acid depletion or supplementation and the effect of glucagon and insulin on the amino acid transport mediated by system A were investigated by determining the uptake of either 2‐amino [1‐14C]isobutyric acid (AIB) or N‐methyl 2‐amino [1‐14C]isobutyric acid (MeAIB) in rat hepatocytes, freshly isolated at different stages of pre‐ and postnatal development. The data obtained show that the Na+‐dependent uptake was higher at the earliest developmental stages, and steadily decreased until the adult level. The hormones increased AIB and MeAIB uptake enhancing the Vmax, while the Km was unchanged. This effect was evident in cells from adult and 18–20‐day‐old fetuses, while no response was present before the 18th day of fetal life and in the perinatal period. Actinomycin D or cycloheximide abolished this hormone‐dependent increase. A decrease in AIB and MeAIB transport after incubation in an amino acid‐rich medium was demonstrated at all ages tested, but was particularly evident in the prenatal life. The increase in the activity of the system following amino acid starvation was shown to be mostly dependent from de novo protein synthesis in the fetal life; on the contrary in the adult the increase appeared to be more linked to the releas from transinhibition of the transport.
Differentiation‐associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells
Authors: Heikkilä Jari E. , Scott Ian G. , Suominen Liisa A. , Åkerman Karl E. O.
Published: February 2005

Muscarinic receptor‐linked increases in intracellular free Ca2+ as measured with quin‐2 and Ca2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH‐SY5Y. Induction of differentiation with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca2+ and stimulation of Ca2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3H‐NMS, by a non‐labelled agonist was studied.
Role of Na‐K ATPase in regulation of resting membrane potential of cultured rat skeletal myotubes
Authors: Brodie Chaya , Bak Asia , Shainberg A. , Sampson S. R.
Published: February 2005

The role of Na‐K ATPase in the determination of resting membrane potential (Em) as a function of extracellular K ion concentration was investigated in cultured rat myotubes. The Em of control myotubes at 37°C varied as a function of (K+)0 with a slope of about 58–60 mV per ten‐fold change in (K+)0. Inhibition of the Na‐K pump with ouabain or by reduced temperature revealed that this relation consists of two components. One, between (K+)0 of 10 and 100 mM, remains unchanged by alterations in enzyme activity; The second, between (K+)0 of 1 and 10 mM, is related to the amount of Na‐K pump activity, the slope decreasing as pump activity decreases. Indeed, with complete inhibition of the Na‐K pump, Em does not change over the range of (K+)0 1 to 10 mM. Measurements of 86Rb efflux and input resistance of individual myotubes showed that membrane permeability does not change as (K+)0 increases from 1 to 10 mM but increases as (K+)0 increases further. Monensin, which increases Na ion permeability, increases Em at values of external K+ below 10 mM, and is without effect at higher values of K+ concentration. The effect of monensin is blocked by ouabain. Tetrodotoxin, which blocks voltage‐dependent Na+ channels, decreases Em at low (2–10 mM) K+. We conclude that changes in Em as a function of extracellular K+ concentration in the physiological range are not adequately explained by the diffusion potential hypothesis of Em, and that other theories (electrogenic pump, surface‐absorption) must be considered.
The effects of sulfhydryl modifying reagents on nonhormonal and hormonally regulated hexose transport in cultured human skin fibroblasts
Authors: Germinario Ralph J. , Vlachopoulou Fotini
Published: February 2005

The effects of various sulfhydryl modifying reagents on hexose transport in cultured human skin fibroblasts were studied. H2O2 was observed to have no effect on 2‐deoxy‐D‐glucose transport in serum‐starved glucose‐fed cells. The elevation of hexose transport rates in cells by glucose deprivation, insulin, or serum stimulation rendered them sensitive to H2O2. Hexose transport in glucose‐deprived cells was inhibited 51–55% by 1–2 mM H2O2, while hexose transport in insulin or serum‐stimulated glucose‐fed cells was inhibited 45% and 46%, respectively. H2O2 inhibition was blocked or reversed by 8 mM dithiothreitol. N‐ethyl‐maleimide (NEM), a permeant, sulfhydryl reagent, elicited effects on hexose transport similar to those effected by H2O2 (i.e., in glucose‐deprived and insulin‐stimulated cells, inhibition of hexose transport was 44% and 23%, respectively). Impermeant sulfhydryl reagents such as dithio(bis)nitrobenzoic acid (DTNB) and N‐iodoacetyl‐N'‐(5‐sulfo‐1‐naphthylethylenediame (1,5,‐I‐AEDANS)) had no inhibitory effect on hexose transport under any conditions (i.e., glucose‐fed, glucose‐deprived, and insulin‐stimulated cells). DTNB and 1,5‐I‐AEDANS afforded no protection from the action of H2O2 on hexose transport. The data suggest that the sensitive sites are thiol in nature and are located at an intramembrane or intracellular site and probably not exofacial.
2‐Mercaptoethanol acts at the restricted stage of interleukin‐2 dependent lymphocyte proliferation
Authors: Sugama Kazushige , Namba Yuziro , Hatanaka Masakazu , Hanaoka Masao
Published: February 2005

An interleukin‐2 (IL‐2) dependent murine cell line (TN‐9) which could be grown continuously with the crude culture supernatant of concanavaline A‐stimulated rat or mouse spleen cells could not synthesize DNA in the culture medium supplemented with partially purified or recombinant IL‐2. The cell growth was restored by adding another factor obtained from the same crude culture supernatant. This factor, physicochemically inseparable from serum albumin, was also obtained from the culture medium added with 2‐mercaptoethanol (2‐ME) and incubated at 37°C for 24 hr without the cells. By the experiments using semi‐synchronized cell population, it was demonstrated that 2‐ME or 2‐ME carrying protein acted at the restricted process(es) of cell proliferation which occurred between IL‐2‐acting stage and the initiation of DNA synthesis.
Coordinate expression of c‐myc, c‐myb, and histone H4 genes in reversibly differentiating HL 60 cells
Authors: Brelvi Zamir S. , Studzinski George P.
Published: February 2005

The expression of oncogenes c‐myc and c‐myb in human leukemic cells HL 60 was compared to the expression of histone H4 gene, known to be cell‐cycle dependent. Steady‐state levels of mRNA transcribed from these genes were determined by simultaneous hybridization of Northern transfers with four probes, and the rates of gene expression were measured by nuclear transcription (“run‐on”) assays. Expression of genes c‐myc, c‐myb and histone H4 varied coordinately and in parallel with the rates of DNA synthesis, while the rates of total and ribosomal RNA synthesis, the expression of gene c‐Haras, unrelated to proliferation of these cells, and gene p 72, a constitutively expressed human gene, were unchanged. Further, the levels of c‐myc and c‐myb mRNA but not p 72 mRNA were higher in cell populations enriched for S phase cells. Thus, transcription of genes c‐myc and c‐myb in HL 60 cells appears to be linked to DNA replication in a manner previously demonstrated for core histone gene expression.
Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: Evidence for a maturation model for the formation of lysosomes
Authors: Roederer Mario , Bowser Robert , Murphy Robert F.
Published: February 2005

The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11°C; acidification was limited to pH 6–6.5 at temperatures between 13°C and 17°C. In contrast, a rapid drop to pH 6–6.5 followed by acidification to pH 5–5.5 was observed at temperatures above 19°C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98:1757–1762). The timing of exposure of material internalized by fluid‐phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37°C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13° and 21°C, the rate of hydrolysis was reduced to 31–44% of that at 37°C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20°C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low‐density organelle containing lysosomal enzymes.
Thrombin chemotactic stimulation of HL‐60 cells: Studies on thrombin responsiveness as a function of differentiation
Authors: Bar‐Shavit R. , Hruska K. A. , Kahn A. J. , Wilner G. D.
Published: February 2005

Thrombin, a major procoagulant enzyme and growth factor, is also selectively chemotactic for monocytes and macrophages but not for neutrophils. This effect stands in contrast to other well‐known chemotactic agents such as fMet‐Leu‐Phe, C5a fragments, and LTB4, which stimulate directed cell movement in both cell types, and have important physiological implications. The human leukemic cell line HL‐60, which is capable of differentiating either along granulocytic or monocytic lineages, was therefore used to explore the development of this selective monocyte/macrophage chemotactic response to thrombin. Esterolytically inactive DIP‐α‐thrombin, as well as the thrombin‐derived chemotactic peptide CB67–129, elicits a dose‐dependent chemotactic response in HL‐60 cells differentiated to monocytelike cells by treatment with 1,25(OH)2D3 (HL‐60/mono), whereas no such response is evident in either undifferentiated HL‐60 cells or in cells differentiated into granulocytes by treatment with DMSO (HL‐60/gran). Similarly, early events which characterize stimulation of inflammatory cells by chemotactic agents are also evident, but only in monocyte‐differentiated cells. In HL‐60/mono, thrombin selectively stimulates rapid cytosolic Ca2+ elevation as well as rapid cytoskeletal association of cytosolic actin. Following thrombin stimulation, maximal actin association in these cells occurs within 30 sec (declining to basal levels at the end of 5 min), and maximal Ca2+ elevations are also evident within 15–20 sec, suggesting a temporal relationship between these two events. Thus, the events accompanying stimulation of HL‐60/mono by thrombin are characteristic of those seen following stimulation of inflammatory cells by chemotaxins, with a major difference being the selectivity of thrombin as a chemotaxin for cells of macrophage/monocytic lineage. The selective chemotactic responsiveness of HL‐60/mono to thrombin appears to relate to the development of specific receptors on these cells as part of monocytic differentiation: HL‐60/mono (but HL‐60/gran nor undifferentiated HL‐60) are capable of significant specific 125‐I‐labeled α‐thrombin‐binding (ka∼20 nM), and possess an estimated 400,000 thrombin‐binding sites per cell. Our finding further suggest that the thrombin response of HL‐60 and particularly the expression of thrombin receptors on these cells may serve as a useful model system for exploring the biology of monocyte/macrophage differentiation.
Distribution of excitatory junction potential amplitude in cat cerebral arteries: Examination of its quantal nature and modulation by opiates
Authors: Madden Jane A. , Kalbfleisch John H. , Harder David R.
Published: February 2005

We used scorpion venom to release small amounts of an excitatory neurotransmitter from adventitial nerves in cat left anterior descending cerebral artery. We used glass microelectrodes to measure and record postsynaptic electrical events of minimal amplitude. These events were similar to postsynaptic spontaneous and electrically evoked excitatory junction potentials (ejp's) seen in skeletal muscle. We performed a frequency analysis of the ejp amplitudes to determine if they fit a unimodal or multimodal distribution. We also investigated the effects of phentolamine, norepinephrine, hydromorphone, and morphine on ejp amplitude and frequency in the artery. Statistical analysis of the ejp frequency and amplitude revealed a multipeaked distribution with decreasing peaks. These results were similar to the distribution reported for acetylcholine release in skeletal muscle. The ejps were inhibited by phentolamine, which suggested that these events were adrenergically mediated. Norepinephrine and the opiates, hydromorphone and morphine, reduced the frequency and amplitude of the ejp's. The vessels also constricted to increasing doses of norepinephrine both under control conditions and in the presence of opiate. These results suggest that norepinephrine blocks the ejp's by a feedback mechanism at the presynaptic membrane and that endorphins and/or enkephalins, also acting at this presynaptic site, may modulate neurotransmission in the cerebral circulation.
Induction of heat shock proteins in Chinese hamster ovary cells and development of thermotolerance by intermediate concentrations of puromycin
Authors: Lee Yong J. , Dewey William C.
Published: February 2005

During 4 hr after puromycin (PUR: 20 μg/ml) treatment, the synthesis of three major heat shock protein families (HSPs: Mr = 110,000, 87,000, and 70,000) was enhanced 1.5‐fold relative to that of untreated cells, as studied by one‐dimensional gel electrophoresis. The increase of unique HSPs, if studied with two dimensional gels, would probably be much greater. In parallel, thermotolerance was observed at 10−3 isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5°C or 43°C, respectively. However, thermotolerance was induced by only intermediate concentrations (3–30 μg/ml) of puromycin that inhibited protein synthesis by 15–80%; a high concentration of PUR (100 μg/ml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01–10 μg/ml) of cycloheximide that inhibited protein synthesis by 5–94%. Furthermore, after PUR (20 μg/ml) treatment, the addition of cycloheximide (CHM: 10 μg/ml), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families.

This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43°C in the presence of CHM or PUR immediately after a 2‐hr pretreatment with CHM or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1–2 hr, and did not have any obvious relationship to the synthesis of HSP families. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after CHM or PUR treatment immediately before and during heating at 43°C, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5°C caused by heating at 43°C after cells were incubated for 2–4 hr following pretreatment with an intermediate concentration of PUR.