bluuebunny/crossref_metadata_2025 huggingface dataset:
Proportional equilibration of K, Na ions, and sucrose molecules in pig lenses incubated in the presence of the non‐ionic detergent triton X‐100
Authors: Miseta A. , Kellermayer M. , Ludany A. , Cameron I. L. , Hazlewood C. F.
Published: February 2005

The release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X‐100 (a non‐ionic detergent). This Triton X‐100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X‐100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein‐water‐ion interactions within the lens is the rate‐limiting step of the observed equilibration of monovalent cations and sucrose molecules.
Structural/functional relationships between internal and external MSH receptors: Modulation of expression in Cloudman melanoma cells by UVB radiation
Authors: Chakraborty Ashok K. , Orlow Seth J. , Bolognia Jean L. , Pawelek John M.
Published: February 2005

Expression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells (Orlow et al: J. Cell. Physiol., 142: 129–136, 1990). Here, we show that internal and external receptors for MSH are of identical molecular weights (50–53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co‐purified with a sub‐cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs (Bologniaet al: J. Invest. Derm., 92:651–656, 1989). Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomittant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH.
The basic fibroblast growth factor‐saporin mitotoxin acts through the basic fibroblast growth factor receptor
Authors: Lappi Douglas A. , Maher Pamela A. , Martineau Darlene , Baird Andrew
Published: February 2005

We have confirmed the hypothesis that a mitotoxin resulting from the conjugation of basic fibroblast growth factor and saporin exerts its cytotoxic effect through specific interaction with the basic fibroblast growth factor (FGF) receptor. Accordingly, the mitotoxin stimulates tyrosine phosphorylation of the 90 kD substrate that characterizes the initial cellular response to basic FGF. Cross‐linking experiments show that radio‐labeled basic fibroblast growth factor‐saporin (FGF‐SAP) binds to the receptor. Suramin, an inhibitor of growth factor receptor binding, inhibits the cytotoxicity of basic FGF‐SAP. In a study of 4 different cell types, there is a decrease in the ED50 of the mitotoxin as the receptor number per cell increases. We have verified the cytotoxicity of the mitotoxin in 3 different assay systems. As expected, it is effective in the inhibition of protein synthesis and DNA synthesis, as well as of cell count. Binding of basic FGF‐SAP which will result in cytotoxicity occurs very rapidly: 5 minutes of incubation of 10 nM basic FGF‐SAP with cells results in 80% inhibition of cell count. The in vitro data indicate that basic FGF‐SAP is a receptor specific and potent suicide antagonist of basic FGF. Its potential as an anti‐FGF for therapeutic and research uses in vivo is discussed.
Steady state levels of hepatic α1 and β2‐adrenergic receptors and gene transcripts during development of the male rat
Authors: Rossby S. Paul , Cornett Lawrence E.
Published: February 2005

Metabolic events stimulated by epinephrine and norepinephrine in hepatocytes isolated from fetal and early postnatal male rats are largely mediated through the β2‐adrenergic receptor‐/cyclic AMP dependent‐system, whereas the same stimuli are transduced through the α1‐adrenergic receptor‐/phosphatidylinositol dependent‐system in hepatocytes isolated from young adult male rats. This developmental transition was investigated by correlating hepatic α1‐ and β2‐adrenergic receptor gene transcript levels with receptor levels as determined with selective radioligands in livers from late fetal to postnatal day 120 male Sprague‐Dawley rats. β2‐Adrenergic receptor concentration, initially high in membrane preparations isolated from fetal livers (203 ± 21 fmol/mg protein), dropped precipitously n postnatal day 6 livers (14± 2 fmol/mg protein) and remained low throughout development out to postnatal day 90.β‐Adrenergic receptor mRNA levels were highest in fetal livers, were decreased somewhat in postnatal day 6 livers and were uncetectable in livers beyond postnatal day 15. In contrast, hepatic α‐adrenergic receptor concentration was relatively low in fetal livers (86 ± 25 fmol/mg protein) and remained low until postnatal day 18. Thereafter, a steady increase in α1‐adrenergic receptors was observed until adult levels. (270 ± 24 fmol/mg protein) were achieved at postnatal day 27. α1‐Adrenergic receptor mRNA levels increased ∼ 3‐fold, reaching a peak at postnatal day 24. Surprisingly, at postnatal day 30 hepatic α1‐adrenergic receptor mRNA levels dropped to fetal levels; but, gradually increased with continued development. Thus, hepatic α1‐ and β2‐adrenergic receptors appear to be under complex regulatory control which may include transcriptional, as well as post‐transcriptional, mechanisms.
Modulation of responsiveness to cAMP stimulating agonists by phorbol ester in fetal rat osteoblasts
Authors: Bos M. P. , van Leeuwen J. P. T. M. , Herrmann‐Erlee M. P. M.
Published: February 2005

We studied the effect of activation of protein kinase C (PKC) by a phorbol ester on cAMP accumulation in fetal rat osteoblasts. Activation of PKC by phorbol 12‐myristate 13‐acetate (PMA) caused a potentiation of cAMP accumulation induced by parathyroid hormone (PTH), forskolin, and cholera toxin. The results suggest that the potentiating effect of PMA on PTH‐induced cAMP accumulation was not due to an effect on the PTH‐receptor nor to an effect on cAMP degradation, as the effect of PMA persisted in the presence of a phosphodiesterase inhibitor. Pretreatment of the cells with pertussis toxin did not prevent the action of PMA, indicating that PMA does not act via the inhibitory G‐protein. PMA had a biphasic effect on prostaglandin E2 (PGE2)‐induced cAMP accumulation; i.e., at concentrations ⩾ 10−6 M, PMA potentiated the PGE2‐induced cAMP response but PMA attenuated cAMP accumulation induced by concentrations of PGE2 ⩽ 5.10−7 M. From our data we conclude that PKC can interact with a stimulated cAMP pathway in a stimulatory and inhibitory manner. Potentiation of cAMP accumulation is probably due to modification of the adenylate cyclase complex, whereas attenuation of stimulated cAMP accumulation appears to be due to an effect on a different site of the cAMP generating pathway, which may be specific to PGE2‐induced cAMP accumulation.
Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas
Authors: Bisgaard Hanne Cathrine , Thorgeirsson Snorri S.
Published: February 2005

The appearance of differentiated hepatocytes in the adult rat pancreas as well as pancreatic‐type tissue in the adult rat liver can be experimentally induced (Reddy et al.: J. Cell Biol., 98:2082–2090, 1984; Rao et al., J. Histochem. Cytochem., 34:197–201, 1986). These observations suggest a lineage relationship between cell compartments present in rat liver and pancreas. The present data demonstrate that epithelial cell lines with almost identical phenotypes can be established from adult rat liver and pancreas. The established cell lines showed similar morphologies as established by light‐and electron‐microscopic studies. The cell lines showed a unique expression pattern of intermediate filament proteins. Vimentin, actin, and β‐tubulin were present in all cell lines. In addition, simple epithelial type II cytokeratins 7 and 8 were found to be coexpressed with the type I cytokeratin 14 in several of the cell lines. Neither the type I cytokeratins 18 and 19, which are the normal partners for cytokeratins 8 and 7 in filament formation, nor the type II cytokeratin 5 could be detected despite the fact that filaments were formed by both cytokeratins 8 and 14. This suggests that cytokeratin 14 acts as an indiscriminate type I cytokeratin in filament formation in the established cell lines. The cell lines expressed the same sets of LDH and aldolase isoenzymes and identical sets of glutathione transferase subunits. In addition, the epithelial cell lines from liver and pancreas were equally sensitive to the growth‐inhibitory effects of TGF‐β1. No expression of tissue‐ or cell‐specific proteins such as α‐fetoprotein, albumin, amylase, elastase, or γ‐glutamyl transpeptidase were detected. The almost identical phenotypes of the hepatic and pancreatic cell lines suggest that they may be derived from a common primitive epithelial cell type present in both rat liver and pancreas. In contrast to parenchymal cells, these cells have an extended capacity for proliferation in vitro and may represent a progeny from a “precursor” or “stem” cell compartment in vivo.
Proliferation of smooth muscle cells in the inner and outer layers of the tunica media of arteries: An in vitro study
Authors: Betz E. , Fallier‐Becker P. , Wolburg‐Buchholz K. , Fotev Z.
Published: February 2005

During the development of atherosclerotic and fibromuscular proliferates/lesions, smooth muscle cells (SMC in the media, particularly near the lumen, are activated to migrate into the intima, where they continue to proliferate to form an intimal thickening. It is to date unclear whether SMCs situated adjacent to the adventitia possess a lower capacity to proliferate because they are a special subpopulation of medial SMCs or because the adventitia excerts an inhibitory effect. We have, therefore, developed an in vitro system whereby we have attempted to clear up this uncertainty. The following observations were made from the in vitro experiments:

Media‐explants from rabbit aorta were laid on a polycarbonate filter with pores 5 μm in diameter. The SMCs migrated through the pores and formed a fibromuscular proliferate on the other side of the filter.

Endothelial cells were seeded on one side of the filter before media‐explants were laid on the other side of the filter. The confluent endothelium inhibited migration of SMCs through the filter pores.

Media‐explants were placed between two polycarbonate filters (pores 5 μm diameter). In this “sandwich” arrangement SMCs migrated through both filters, i.e., in both directions. The quantity of migrating and proliferating cells through both filters was almost identical. This suggests that there is no difference in the migratory and proliferate capacity of SMCs in the inner and outer layers in the media of arteries.

To investigate the influence of the adventitia on medial SMCs, media‐explants were placed between a lower (5 μm) and an upper (0.2 μm) filter adventitia‐explants were laid above the media‐explants. The 0.2 μm filter prevented migration of SMCs from the media‐explant into the adventitia and migration of fibroblasts from the adventitia into the media. Interestingly, the adventitial tissue inhibited proliferation of SMCs at the abluminal and migration and proliferation at the luminal side of the media‐explant; the number of cells migrating through the 5 μm pores at the luminal side was diminished, suggesting that the adventitial tissue has an antiproliferative influence on SMCs. Moreover, it was found that in media‐explants near the filter with adventitia, the medial SMCs were in a better preserved condition than at the de‐endothelialised luminal side.

As a control, cultures consisting of media‐explants were incubated without filters (i.e., explant organ cultures). The proliferates in the concavity (luminal side) exhibited a pattern of proliferating SMCs different from that of the cells at the abluminal convexity. This finding indicates that the different texture of vessel walls near the adventitia and near the lumen may also influence cell migration and proliferation.
Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet‐derived growth factor in osteoblast‐enriched cultures from fetal rat bone
Authors: Centrella Michael , McCarthy Thomas L. , Kusmik William F. , Canalis Ernesto
Published: February 2005

Platelet‐derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF‐A or PDGF‐B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF‐BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systemically in vivo, has not been reported. Therefore recombinant human PDGF‐BB, PDGF‐AB, and PDGF‐AA were evaluated in osteoblast‐enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half‐maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF‐BB, PDGF‐AB, and PDGF‐AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF‐BB being the most inhibitory. Binding studies with 125I‐PDGF‐BB or 125I‐PDGF‐AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I‐PDGF‐BB binding was preferentially displaced by PDGF‐BB (Ki ≈0.7 nM), less by PDGF‐AB (Ki ≈2.3 nM) and poorly by PDGF‐AA. In contrast, 125I‐PDGF‐AA binding was measurably reduced by PDGF‐AA (Ki ≈4.0 nM), but was more effectively displaced by PDGF‐BB or PDGF‐AB (each with Ki ≈0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF‐B subunit binding preferentially mediate these results in osteoblast‐enriched bone cell cultures.
Characteristics of taurine transport in rat liver lysosomes
Authors: Vadgama Jaydutt V. , Chang Kent , Kopple Joel D. , Idriss John‐Michael , Jonas Adam J.
Published: February 2005

Taurine (2‐aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety of cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. Intralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na+‐independent, with an overshoot between 5–10 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km1= 31 ± 1.82 and Km2> 198 ± 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a t1/2 of 5 minutes at 37°C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane β‐alanine carrier system, with inhibition by β‐alanine, hypotaurine, and taurine. α‐Alanine, 2‐methylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP‐taurine [N‐(4‐azido‐2‐nitrophenyl)‐2‐aminoethanesulfonate]. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K+gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.
Hydrocortisone in culture protects the blast cells in acute myeloblastic leukemia from the lethal effects of cytosine arabinoside
Authors: Yang G. S. , Wang C. , Minkin Salomon , Minden M. D. , McCulloch Ernest A.
Published: February 2005

The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. We report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara‐C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara‐C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritlated thymidine (3HTdR) “suicide” technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara‐C concentrations gave very similar dose‐response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara‐C did not lose colony‐forming ability even though the same population was sensitive to 3HTdR. The hydrocortisone effect was dose and time related; protection from ara‐C increased from 10−8 to 10−5 M and was seen after 4 hr exposure but required 8 hr to reach a maximum. We conclude that hydrocortisone can protect blasts from the lethal effects of ara‐C even while the cells are in active DNA synthesis.
Direct evidence for spatial and temporal regulation of transforming growth factor β1 expression during cutaneous wound healing
Authors: Kane Cynthia J. M. , Hebda Patricia A. , Mansbridge Jonathan N. , Hanawalt Philip C.
Published: February 2005

The expression of transforming growth factor (TGFβ1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti‐CC and anti‐LC) following cutaneous injury. The anti‐LC antibody binds intracellular TGFβ1 constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti‐CC antibody does not react with the form of TGFβ1 present in normal skin as previously shown. TGFβ1 may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGFβ1 protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness porcine skin. Anti‐CC reactive TGFβ1 in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGFβ1 protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGβ1 activity. One day post‐injury anti‐CC reactive TGFβ1 is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post‐injury anti‐CC reactive TGFβ1 is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGFβ1 from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGFβ1 activity. Anti‐CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti‐LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti‐LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti‐CC reactive TGFβ1 disappears while constitutive anti‐LC reactive TGFβ1 persists. Thus, changes in the structure or conformation of TGFβ1, its localization, and perhaps its activity vary in a spatial and temporal manner following cutaneous injury and correlate with physiological changes during wound healing.
Establishment of a variety of human bone marrow stromal cell lines by the recombinant SV40‐adenovirus vector
Authors: Aizawa Shin , Yaguchi Makoto , Nakano Masaru , Inokuchi Sadaki , Handa Hiroshi , Toyama Keisuke
Published: February 2005

Various human bone marrow stromal cell lines were established from the adherent cell populations by introduction of the recombinant SV40‐adenovirus vector with an infection or electric poration procedure. As compared with DMA transfection, the vector introduction was able to immortalize the cells with more than 100 times higher efficiency. Morphological and cytochemical analyses indicated that various cloned cell lines with different properties were isolated by the vector introduction. All the established cell lines expressed SV40 large T antigen. These results provided the evidence indicating that the recombinant SV40‐adenovirus vector was a useful tool to establish a variety of cell lines with different biological activities from human bone marrow stroma.
Effects of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro
Authors: Migliaccio Giovanni , Migliaccio Anna Rita , Druzin Maurice L. , Giardina Patricia‐Jane V. , Zsebo Krisztina M. , Adamson John W.
Published: February 2005

We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony‐stimulating activity under fetal bovine serum (FBS)‐deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony‐stimulating factor (GM‐CSF), and interleukin 3 (IL‐3) to stimulate colony growth. Colony morphology was determined by the late‐acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL‐3 and Epo resulted in the largest number of mixed‐cell colonies‐ a larger number than observed with IL‐3 and Epo alone even in FBS‐supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed‐cell colony‐forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date.
Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells
Authors: Raub Thomas J. , Newton Carolyn R.
Published: February 2005

Primary culture of bovine brain microvessel endothelial cells (BMECs) were used to examine the cycling kinetics of ferrotransferrin (Tf) and to provide evidence for a transcytotic pathway in vitro. Binding of 125I‐Tf to BMECs grown on matrix‐coated plastic was measured in the presence of saponin to calculate the total number of transferrin receptors (TfRs). Nonlinear regression analysis of the binding isotherm showed that there were 100,000 high‐affinity receptors per cell and that expression was maximum at cell confluence. Binding of Tf at 4°C indicated that there was a large intracellular receptor pool comprising 85–90% of the total cellular receptors. Accumulation of Tf at 37°C, inhibited at low temperature and in the presence of metabolic poisons, occurred with an initial rate coefficient of 0.030 min−1 and this decreased by 83% after 60 min. Concomitant accumulation of 59Fe from Tf‐59Fe was linear. In the absence of externally added ligand, 80% of the accumulated 125I‐Tf was released into the medium with a rate coefficient of 0.017 min−1 and this was inhibited at low temperature. In the presence of the weak base primaquine, the accumulation of Tf and 59Fe and the efflux of Tf were decreased. Moreover, phorbol myristate acetate (PMA) caused a 30% increase in surface TfRs and an 82% increase in Tf accumulation, although the size of the recycling pool remained unchanged. Despite the low numbers of TfR expressed by post‐confluent cells, filter‐grown BMEC monolayers were used to measure transcytosis of Tf. A small portion of the Tf that was accumulated from the apical side entered a transcytotic pathway. Most of the Tf and all of an accumulated fluid‐phase tracer were recycled towards the apical side. These results showed that cultured BMECs cycle Tf‐TfR complexes slowly and vectorially and suggested that the large intracellular receptor pool may facilitate steady state accumulation and regulate transcellular transport of iron.
Dexamethasone and retinoic acid regulate the expression of epidermal growth factor receptor mRNA by distinct mechanisms
Authors: Oberg Kerby C. , Carpenter Graham
Published: February 2005

Retinoic acid and dexamethasone have antagonistic effects on epidermal growth factor (EGF) receptor expression in fetal rat lung (FRL) cells: Receptor synthesis is enhanced by retinoic acid and reduced by dexamethasone. In the presence of actinomycin D, neither agent has the capacity to modify receptor synthesis or 125I‐EGF binding capacity. Northern blot analysis demonstrates a tenfold increase in EGF mRNA following retinoic acid treatment and a 60% decrease in receptor message levels after dexamethasone treatment. To dissect the mechanisms of these effects, the expression of mRNA was separated from effects requiring protein synthesis by the use of cycloheximide and actinomycin D. Ligand binding, EGF receptor protein synthesis, and mRNA levels were measured in cultures of FRL cells that were incubated with retinoic acid or dexamethasone in the presence of cycloheximide, then washed and reincubated with fresh media containing actinomycin D, but not retinoic acid, dexamethasone, or cycloheximide. The results demonstrate that dexamethasone reduces the expression of EGF receptor mRNA in the absence of protein synthesis. In contrast, the mechanism by which retinoic acid increases the expression of EGF receptor mRNA requires protein synthesis. These data indicate that, in FRL cells, dexamethasone negatively regulates EGF receptor mRNA in a direct manner, while retinoic acid controls transcription of an intermediate protein, possibly a transcription factor, that subsequently increases transcription of receptor message.
Transport of lactate in Plasmodium falciparum‐infected human erythrocytes
Authors: Kanaani Jamil , Ginsburg Hagi
Published: February 2005

The intraerythrocytic human malarial parasite Plasmodium falciparum produces lactate at a rate that exceeds the maximal capacity of the normal red cell membrane to transport lactate. In order to establish how the infected cell removes this excess lactate, the transport of lactate across the host cell and the parasite membranes has been investigated. Transport of radiolabeled L‐lactate across the host cell membrane was shown to increase ca. 600‐fold compared to uninfected erythrocytes. It showed no saturation with [L‐lactate] and was inhibited by inhibitors of the monocarboxylate carrier, cinnamic acid derivatives (CADs), but not by the SH‐reagent p‐chloromercuriphenyl sulfonic acid (PCMBS). These resultes suggest that L‐lactate is translocated through CAD‐inhibitable new pathways induced in the host cell membrane by parasite activity, probably by diffusion of the acid form and through a modified native monocarboxylate:H+ symporter. Continuous monitoring of extracellular pH changes occurring upon suspension of infected cells in isoosmotic Na‐lactate solutions indicates that part of the lactate egress is mediated bty anionic exchange through the constitutive, but modified, anion exchanger. The transport of L‐lactate across the parasite membrane is rapid, nonsaturating, and insensitive to either CADs or PCMBS, or to the presence of pyruvate. L‐lactate uptake increased transiently when external pH was lowered and decreased when ΔpH was dissipated by the protonophore carbonylcanide m‐chlorophenyl hydrazone (CCCP). These results are compatible with L‐lactate crossing the parasite membrane either as the undissociated acid or by means of a novel type of lactate −/H+ symport.
Germ cell mitogenic activity is associated with nerve growth factor‐like protein(s)
Authors: Onoda M. , Pflug B. , Djakiew D.
Published: February 2005

The mitogenicity of germ cell proteins released from round spermatids (RS) and pachytene spermatocytes (PS) was investigated. Germ cells were isolated by centrifugal elutriation from 90‐day‐old rat testes and incubated in a supplement enriched culture media that lacked exogenous proteins. The conditioned culture media of RS and PS were dialysed/concentrated and lyophilized to prepare RS protein (RSP) and PS protein (PSP). Mitogenic activity of RSP and PSP was determined by 3H‐thymidine incorporation into Swiss 3T3 fibroblasts. RSP and PSP stimulated 3H‐thymidine incorporation by fibroblasts in a dose‐dependent manner. At a higher concentration of RSP (300 μg/ml), fibroblast proliferation was stimulated from 6‐ to 20‐fold of control cultures, whereas PSP (300 μg/ml) stimulated fibroblast proliferation 2.5‐fold of control cultures. Since RSP exhibited substantially greater mitogenic activity than PSP we further investigated the RSP mitogenic substance(s) by immunoneutralization with antibodies against several growth factors. The mitogenic activity of RSP was significantly reduced by treatment with nerve growth factor (NGF) antibody, while neither the treatment of RSP with acidic fibroblast growth factor (aFGF) antibody, nor basic fibroblast growth factor (bFGF) antibody significantly modified the mitogenic activity of RSP. Interestingly, murine NGF‐β, recombinant human NGF‐β, and bovine serum albumin (BSA) did not exhibit mitogenic activity on 3T3 fibroblasts. Nevertheless, the presence of a NGF‐like protein in RS and PS was confirmed by indirect immunofluorescence staining with a murine NGF antibody. Subsequently, a Western blot analysis with the NGF antibody identified two immunoreactive bands of 41 ± 2 kDa and 51 ± 1 kDa in both RSP and PSP under reduced conditions. These germ cell NGF‐like proteins were apparently different from similarly prepared murine and human NGFs (13 kDa) in their molecular weight. Furthermore, neurite outgrowth from pheochromocytoma cells (PC‐12), a functional bioassay for NGF‐like activity, was stimulated by addition of RSP and PSP to the culture media of the PC‐12 cells. These results demonstrate mitogenic activity in germ cell proteins (RSP and PSP) and identify a NGF‐like protein(s) which is associated with most of this activity.
Thrombin inhibits proliferation of the human megakaryoblastic MEG‐01 cell line: A possible involvement of a cyclic‐AMP dependent mechanism
Authors: Vittet Daniel , Mathieu Marie‐Noëlle , Launay Jean‐Marie , Chevillard Claude
Published: February 2005

Thrombin, a potent platelet activating agent, has previously been found to increase intracellular calcium levels and/or thromboxane A2 synthesis in leukemic cell lines exhibiting specific markers of the megakaryocyte/platelet lineage. However, its functional role on these cells has not been defined. As thrombin is implicated in the regulation of cellular proliferation or differentiation in various other cell types, we investigated the functional effects of thrombin on the megakaryoblastic MEG‐01 cell line, and further explored its receptor coupling mechanisms on these cells. We observed that thrombin caused in 1% serum containing culture medium, a reduction in the proliferation of MEG‐01 cells, without affecting their differentiation stage as determined by the expression of platelet glycoproteins GPIIb/IIIa and GPIb, FVIII‐related‐antigen and cell‐size measurement, which are specific markers for megakaryocyte maturation. In increases in cAMP levels, and in inositol‐trisphosphate formation and intracellular Ca2+ levels. All these responses required thrombin proteolytic activity. The lipoxygenase inhibitor, nordihydroguaiaretic acid, blunted thrombin‐induced calcium increase without affecting thrombin‐induced increase in cAMP levels, suggesting different thrombin coupling mechanisms with these two second messenger pathways. In addition, the inhibitory effect of thrombin on MEG‐01 cell growth was mimicked by cAMP level enhancing agents such as forskolin, prostaglandin E1 and Bt2 cAMP. These results suggest the involvement of a cAMP‐dependent mechanism in the thrombin‐induced reduction in MEG‐01 cell growth.
Growth and function of primary rabbit kidney proximal tubule cells in glucose‐free serum‐free medium
Authors: Jung Jee Chang , Lee Sang‐Mog , Kadakia Nina , Taub Mary
Published: February 2005

The properties of primary rabbit kidney proximal tubule cells in glucose‐free serum‐free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose‐free and in glucose‐supplemented medium. Growth in glucose‐free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose‐free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose‐free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D‐glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.
Differential induction of the c‐fos promoter through distinct PDGF receptor‐mediated signaling pathways
Authors: Salhany Kevin E. , Robinson‐Benion Cheryl , Candia Albert F. , Pledger W. J. , Holt Jeffrey T.
Published: February 2005

The multiple isoforms of PDGF induce fibroblastic mitogenesis through two distinct PDGF receptors, α and β. The molecular mechanisms by which these α and β PDGF receptors regulate gene expression are poorly understood. We present data which indicates that differential induction of c‐fos gene expression by PDGF isoforms occurs through distinct PDGF α and β receptor‐mediated signaling pathways. Comparison of PDGF‐AA with PDGF‐BB stimulation showed that PDGF‐BB induced prolonged expression of the c‐fos gene in BALB/c‐3T3 cells, but that PDGF‐AA induced more potent activation of the serum response element (SRE) in transient transfection assays. PDGF‐AA, which binds α but not β PDGF receptors, could only induce the SRE through a protein kinase C (PKC)‐dependent pathway, whereas PDGF‐BB, which binds both α and β PDGF receptors, could also induce the SRE through a PKC‐independent pathway. These results suggest that PDGF α receptors activate the PKC‐dependent signaling pathway while PDGF β receptors also activate a PKC‐independent pathway. In addition, we found that PDGF‐BB could induce another c‐fos promoter element within the — 90 to + 10 region, suggesting that the more potent mitogenic effect and prolonged c‐fos gene expression induced by PDGF‐BB may result from cooperativity between more than one c‐fos promoter elements.
TGF‐β blocks early but not late differentiation‐specific gene expression and morphologic differentiation of 3T3 T proadipocytes
Authors: Sparks Rodney L. , Allen Blake J. , Strauss Ethan E.
Published: February 2005

Transforming growth factor‐β (TGF‐β) inhibits morphologic differentiation of BALB/c 3T3 T cells as well as other proadipocyte models. Our prior studies suggested that TGF‐β may act only during the early stages of differentiation induction. However, we did not determine whether TGF‐β was differentially effecting expression of any of the various differentiation‐specific genes or if it could cause down‐regulation of these genes in differentiated cells. Therefore, in the current study we tested the effects of exogenous TGF‐β (0.01–5.0 ng/ml) on morphologic differentiation and on differentiation‐dependent gene expression (Northern and slot blot analyses) at various times during differentiation. When induced to differentiate, 3T3 T cells first undergo predifferentiation growth arrest and from this state molecular, biochemical, and morphological differentiation proceeds. Here it was found that when added prior to the onset of differentiation, TGF‐β was a potent inhibitor of morphologic differentiation as well as of the expression of differentiation‐specific genes such as lipoprotein lipase (LPL) and glycerol‐3‐phosphate dehydrogenase (GPD). However, once morphologic differentiation began, TGF‐β was ineffective in blocking differentiation. In addition, exposure of fully differentiated cells to TGF‐β for up to 72 hours caused no decrease of differentiation‐specific genes and even a 7‐day treatment caused no morphologic dedifferentiation. Tumor necrosis factor also had no detectable effect on fully differentiated cells.
Cyclic AMP potentiates bFGF‐induced neurite outgrowth in PC12 cells
Authors: Ho Paulo Lee , Raw Isaias
Published: February 2005

We report here that basic fibroblast growth factor (bFGF)‐elicited neurite outgrowth in PC12 cells is potentiated by dibutyryl cyclic adenosine monophosphate (dbcAMP) or forskolin. This property was also described for nerve growth factor (NGF), suggesting that both NGF and bFGF may share common intracellular events leading to neurite outgrowth and synergism with dbcAMP and forskolin. The synergistic effect of dbcAMP and forskolin is specific, since treatment of PC12 cells with bFGF and dibutyryl cyclic guanosine monophosphate (dbcGMP) or phorbol ester did not change the neurite outgrowth response of cells treated with bFGF alone. Furthermore, neurite outgrowth depends on cellular adhesion. Increasing adhesion by plate treatment with poly‐d‐lysine increases the neurite outgrowth elicited by bFGF alone or bFGF plus dbcAMP. On the other hand, decreasing cellular adhesiveness by plating PC12 cells in semi‐solid agarose renders the cells unable to develop neuritic processes. In addition, 3H‐methylthymidine incorporation studies showed that bFGF‐treated PC12 cells cease growth only when they become fully differentiated after 3–5 days of treatment. In contrast, dbcAMP, which is a poor differentiation factor, is able to block cellular growth after 24 hour treatment. These results suggest that when PC12 cells become differentiated, they stop growing. However, growth inhibition does not necessarily lead to differentiation.
Zinc transport into endothelial cells is a facilitated process
Authors: Bobilya Dennis J. , Briske‐Anderson Mary , Reeves Philip G.
Published: February 2005

The kinetics of zinc transport were examined by measuring the uptake of 65Zn into cultured endothelial cells. This served as a suitable model for characterizing the transport of zinc across a biological membrane (i.e., the plasma membrane). The transport process was saturable under physiological conditions, which indicates a facilitating transport mechanism. Within the physiological range of zinc concentrations, the maximum zinc transport rate was 27 pmoles zinc/(min × mg protein) and it was half maximal at 4.1 μM zinc. Cadmium competitively inhibited zinc transport (Ki = 6.5 μM), while equimolar concentrations of copper and manganese were ineffectual. The rate of zinc transport was substantially reduced at lower temperatures and in the presence of sulfhydryl blockers (sodium iodoacetate and N‐ethylmaleimide). Inhibitors of energy metabolism (2,4‐dinitrophenol and sodium azide) failed to disrupt zinc transport. These results demonstrate that zinc transport into endothelial cells is a facilitated process (i.e., it is carrier mediated and energy‐independent). © 1992 Wiley‐Liss, Inc.
Distinct effects of phorbol esters and exogenous diacylglycerols in the induction of murine thymocyte proliferation
Authors: Stoeck Michael , Howe Rawleigh C. , MacDonald H. Robson
Published: February 2005

Murine thymocytes were stimulated with the protein kinase C activating agents Phorbol‐12‐myristate‐13‐acetate (PMA) or a more physiological membrane permeant diacylglycerol (dioctanoyl‐sn‐glycerol, DiC8) in the presence or absence of exogenous lymphokines (rlL‐1β, rlL‐2). Whereas PMA directly induced reactivity to rlL‐2, DiC8 did not but had to synergize with the calcium ionophore lonomycin. Expression of the p55 chain of the IL‐2 receptor behaved similarly. In the absence of exogenous rlL‐2, thymocytes proliferated in response to a combination of lonomycin and PMA; however, replacing PMA by a single addition of DiC8 did not result in proliferation. Stimulation with lonomycin plus repeated addition of DiC8 induced a low level of thymocyte proliferation and further addition of rIL‐1β resulted in a significant increase. Purified immature (L3T4−Lyt2−) thymocytes behaved similarly, but showed an increased sensitivity to rIL‐1β. Taken together, the data support the idea that PMA and the more physiological diacylglycerols do not possess totally equivalent activities in lymphocyte stimulation. © 1992 Wiley‐Liss, Inc.
Growth and motility of microvascular endothelium are modulated by the relative concentration of gangliosides in the medium
Authors: Alessandri G. , de Cristan G. , Ziche M. , Cappa A. P. M. , Gullino P. M.
Published: February 2005

The data reported were obtained as an attempt to understand whether the change in total concentration and relative ratios of the 3 major corneal gangliosides (GM3, GM2, GD3) previously observed in corneas stimulated by an angiogenic molecule (Ziche et al., 1989) was a relevant event in the angiogenic response of the tissue. The effect on endothelial cell growth was tested for the 3 corneal gangliosides added singly to the culture medium, and GM3 was found to possess a substantial growth inhibitory effect as compared to GM2 and GD3. The growthlimiting effect of GM3 was counteracted to a different degree by the addition of a second ganglioside to the culture medium. A mixture of GM3 + GM2 + GD3 in the proportion similar to that found in the cornea stimulated by an angiogenic molecule was able to sustain a sharp increment in cell growth and motility when added to cultures of capillary endothelium. On the contrary, when the 3 components of the mixture were in the proportion present in the normal cornea, the increment in growth or motility did not occur. A simple change in the relative proportions of the 3 gangliosides was sufficient to trigger or prevent an increment in growth and motility of the endothelial cells. These data in vitro suggest that the changes in concentration and relative ratios of the 3 major corneal gangliosides observed in vivo when the cornea was stimulated by an angiogenic molecule were an event targeted to favour growth and mobilization of the capillary endothelium located within the limbal vessels at the periphery of the cornea. © 1992 Wiley‐Liss, Inc.
Inhibition of heat shock gene expression does not block the development of thermotolerance
Authors: Bader Stephen B. , Price Brendan D. , Mannheim‐Rodman Laura A. , Calderwood Stuart K.
Published: February 2005

After cells have been exposed to a nonlethal heat shock, they develop an enhanced resistance to subsequent prolonged heat shock. This process, termed thermotolerance, correlates with the expression of a group of proteins called the heat shock proteins. When cells are exposed to heat, protein synthesis is rapidly turned off and takes 5–6 hr to recover. In thermotolerant cells, protein synthesis is not blocked by heat. The heat shock proteins are thought to be responsible for the development of thermotolerance and the protection of the protein synthesis machinery from heat inactivation. To test the hypothesis that the heat shock proteins are involved in the heat shock response, we used two inhibitors to block their transcription and expression during heating and then monitored the effect on the development of thermotolerance and on protein synthesis. Camptothecin inhibits DNA topoisomerase I and blocks transcription of all actively transcribed genes, whereas dichloro‐D‐ribofuranosylbenzimidazole (DRB) inhibits only those genes transcribed by RNA polymerase II. Both DRB and camptothecin blocked the heat‐induced expression of the heat shock proteins, but the absence of these proteins did not block either the development of thermotolerance or the protection of protein synthesis after heating. The data indicate that thermotolerance can develop in the absence of new protein synthesis. © 1992 Wiley‐Liss, Inc.