bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnesium‐Dependent stimulation of protein synthesis by the insulin mimic, pervanadate
Authors: Barnes David M. , Sykes Destiny B. , Smith James J. , Miller David S.
Published: February 2005

The insulin mimic, peroxide of vanadate (pervanadate), stimulated 35S‐methionine incorporation into Xenopus oocyte protein in a Mg2+‐dependent manner. Reducing the extracellular Mg2+ concentration from 1.0 to 0.1 mM decreased the pervanadate‐stimulated component of incorporation by 35%; with 0.01 mM Mg2+ or lower, the pervanadate‐stimulated component was abolished. In addition, reducing extracellular Mg2+ to 0.01 mM inhibited about 50% of the insulinstimulated component of methionine incorporation. Mg2+ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn2+ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg2+. Reducing extracellular Ca2+ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 m̈I droplet of buffer containing the Mg2+ indicator dye, mag‐fura‐2, and pervanadate, apparent droplet Mg2+ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg2+. In contrast, apparent extracellular Mg2+ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg2+, but not Ca2+, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg2+, and this change in membrane transport may be an early event in signalling the increase in translation. © 1995 Wiley‐Liss, Inc.
Effect of platelet‐derived growth factor on DNA synthesis and gene expression in bone marrow stromal cells derived from adult and old rats
Authors: Tanaka Hiroshi , Liang C. Tony
Published: February 2005

The effects of platelet‐derived growth factor (PDGF) on DNA synthesis and mRNA expression of osteoblast markers in marrow stromal cells derived from adult (6 months) and old (24 months) rats were examined. Treatment of stromal cells from adult rats with dexamethasone induced the appearance of osteoblast‐like cells. PDGF partially also inhibited the differentiation of stromal cells induced by dexamethasone. In cultures of serum‐starved stromal cells, PDGF stimulated [3H]‐thymidine incorporation into DNA in a dose‐dependent manner with a maximum stimulation of 15‐fold at 500 ng/ml. By comparison, insulin‐like growth factor (IGF‐I) has a small effect on [3H] ‐thymidine incorporation. The effect of PDGF and IGF‐I on DNA synthesis was additive. Treatment of the confluent stromal cells from adult rats with PDGF increased the mRNA level of osteopontin fourfold without any significant effect on alkaline phosphatase and type I collagen mRNAs. In contrast, dexamethasone stimulated the mRNA expression of alkaline phosphatase, type I collagen, and osteopontin 2.1‐, 2.3‐, and 14‐fold, respectively. Addition of PDGF to dexamethasone‐treated cells failed to induce any further increase in osteopontin expression whereas the expression of alkaline phosphatase and type I collagen was partially reduced. The expression of osteocalcin mRNA was negligible in stromal cells but stimulated several fold by dexamethasone and 1,25(OH)2D3. PDGF inhibited drastically the elevation of osteocalcin mRNA. In contrast, IGF‐I stimulated type I collagen expression 100% without any appreciable effect on the expression of osteopontin and alkaline phosphatase. The stimulatory effect of PDGF on osteopontin expression was augmented by IGF‐I. Furthermore, PDGF attenuated the stimulatory effect of IGF‐I on type I collagen expression. The responses of cultured cells from old rats to growth factors were also examined. PDGF or PDGF plus IGF‐I increased [3H]‐thymidine incorporation in stromal cells from old rats but to a lesser extent. However, PDGF was equally effective in stimulating osteopontin expression in cells from both adult and old rats. We concluded that PDGF is a potent mitogen but that the response of stromal cells from old rats is impaired. In addition, PDGF stimulates osteopontin expression in stromal cells and this effect is not age dependent. © 1995 Wiley‐Liss, Inc.
Heat‐Induced bFGF gene expression in the absence of heat shock element correlates with enhanced AP‐1 binding activity
Authors: Erdös Géza , Lee Yong J. , Cho Joong M. , Corry Peter M.
Published: February 2005

Basic fibroblast growth factor (bFGF) has been shown to be a potent mitogen and a promoter of angiogenesis. It has been hypothesized that the expression of the bFGF gene may be induced by stress of various types. To test that hypothesis, we investigated the expression of the bFGF gene during heat treatment in adriamycin‐resistant (MCF‐7/ADR) and‐sensitive (MCF‐7) human breast carcinoma cells. Under normal growth conditions, the bFGF mRNA was detected in MCF‐7/ADR cells, while it was not detectable in MCF‐7 cells by Northern blot analysis. During heating at 41°C, the level of bFGF mRNA increased in MCF‐7/ADR cells and the message became detectable in the MCF‐7 cell line. However, after continuous heating at 41°C for 24 h, the bFGF mRNA level decreased to control level in MCF‐7/ADR cells. Interestingly, simultaneous treatment with heat and 60 m̈g/ml H‐7 (1‐(isoquinolinylsulfonyl)‐2‐methylpiperzine, a potent PKC inhibitor) decreased the level of bFGF mRNA in MCF‐7/ADR cells. These results suggest that a protein kinase, likely PKC, is involved in the transcriptional regulation of the heat‐enhanced bFGF gene expression in human breast carcinoma cells. Although no heat shock element can be identified in the promoter of the bFGF gene, we observed that the AP‐1 binding activity to a TPA responsive element (TRE)‐like sequence in the promoter of bFGF gene was enhanced by heat, as tested by mobility shift assay. Antibody developed against the c‐Jun and c‐Fos proteins inhibited the AP‐1 binding activity to TRE. Therefore, the AP‐1 complex appears to be responsible for the heat‐enhanced binding to the TRE‐like motif of the bFGF gene. Furthermore, the increased AP‐1 binding activity does not require new protein synthesis but activation of the preexisting c‐Jun proteins. © 1995 Wiley‐Liss, Inc.
Regulation of platelet‐derived growth factor A and B chain gene expression in bone marrow stromal cells
Authors: Abboud Sherry L.
Published: February 2005

MBA‐2, bone marrow‐derived endothelial stromal cells, express platelet‐derived growth factor (PDGF) A and B chain mRNAs and secrete PDGF activity that is induced by TGF‐beta. Either chain of the PDGF molecule could modulate hematopoiesis and stromal cell growth. Intracellular pathways that regulate PDGF expression in the marrow microenvironment are unknown. In the present study, we examined the mechanisms that mediate PDGF A and B chain mRNA induction by TGF‐beta and the role of protein kinase C (PKC) and cyclic AMP in PDGF regulation. TGF‐beta was tested in parallel with PMA, an activator of phorbol ester‐dependent PKC isoforms. Both PMA (10−7M) and TGF‐beta (2.5 ng/ml) increased PDGF A and B chain mRNA levels. The serine/threonine protein kinase inhibitor, H7, blocked PDGF A and B chain mRNA induction in response to TGF‐beta. However, down‐regulation of PKC by prolonged incubation with PMA failed to abolish TGF‐beta induction of PDGF A and B chain mRNAs. These findings indicate that induction of PDGF A and B chain mRNAs can be mediated via phorbol ester‐dependent PKC pathway. In contrast, H7‐sensitive protein kinase(s) other than phorbol ester‐sensitive protein kinase C mediate the effect of TGF‐beta. Agents that increase cAMP were also tested for their effect on PDGF gene expression. TGF‐beta‐mediated induction of PDGF A and B chain mRNAs was markedly inhibited by cAMP. cAMP also blocked stimulation of PDGF A chain mRNA by PMA. The positive and negative signaling mechanisms involved in modulating PDGF in the microenvironment may be important for determining hematopoietic and stromal cell responses in vivo. © 1995 Wiley‐Liss, Inc.
Binding and endocytosis of 39 kDa protein by mdbk cells
Authors: Vettenranta Kim , Bu Guojun , Schwartz Alan L.
Published: February 2005

A 39 kDa protein copurifies with the low density lipoprotein receptor‐related protein (LRP) and regulates ligand interactions with LRP. In our recent studies on the clearance of the 39 kDa protein in vivo, we demonstrated that once the liver LRP receptors were saturated, the kidney necame the major organ responsible for the 39 kDa protein clearance (Warshawsky et al., 1993, J. Clin. Invest., 92:937‐944). The current study was undertaken in order to investigate the potential binding and cellular processing of the 39 kDa protein by kidney‐derived MDBK cells. Herein we demonstrate specific, high‐affinity, saturable, and Ca2+‐dependent binding of the 125I‐39 kDa protein to MDBK cells (Kd ∼ 10‐15 nM, 50‐70,000 bindings sites per cell). Cellular uptake and degradation of the 125I‐39 kDa protein by MDBK cells was also demonstrated with kinetics typical of receptor‐mediated endocytosis. Using chemical crosslinking we show that LRP in part mediates the binding of 125I‐39 kDa protein to the MDBK cell surface. In addition, the presence of functional LRP on the MDBK cell surface was confirmed by the specific binding of activated α2‐macroglobulin, another ligand of LRP. Our data thus demonstrate the ability of kidney‐derived MDBK cells to specifically bind, endocytose, and degrade the 39 kDa protein. © 1995 Wiley‐Liss, Inc.
Functional characterization of lymphoid cells generated in serum‐deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice
Authors: Ashany Dalit , Elkon Keith B. , Migliaccio Giovanni , Migliaccio Anna Rita
Published: February 2005

We have investigated the phenotypic and functional characteristics of murine pre‐B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL‐7). Both serum‐supplemented and serum‐deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/Ipr and its congenic strain MRL/+. SCF (100 ng/ml) and IL‐7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum‐supplemented and serum‐deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum‐supplemented cultures but were either ineffective or had modest activity in serum‐deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL‐7 in the absence of serum had almost exclusively a pre‐B cell phenotype (BP‐1+, B220+, slg‐, CD4‐, CD8‐, Mac‐1, RB‐6‐). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12–14. At this time, the pre‐B cells failed to differentiate further and started to die. Pre‐B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 × 106 pre‐B cells had readily detectable serum levels of IgM (54 ± 26 m̈g/ml) and IgG (60 ± 95 m̈g/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (>80% BP‐1+) populations of functional pre‐B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum‐deprived cultures stimulated with SCF and IL‐7. These cultures, therefore, provide a highly enriched source of pre‐B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. © 1995 Wiley‐Liss, Inc.
Evidence for the presence of a Hg‐inhibitable water‐permeability pathway and aquaporin 1 in A5 salivary epithelial cells
Authors: Lazowski Krzysztof W. , Li Jun , Delporte Christine , Baum Bruce J.
Published: February 2005

The molecular basis of water‐permeability in salivary and other exocrine glands is not understood. We have examined two well‐studied salivary epithelial cell lines for evidence of a Hg‐inhibitable water‐permeability pathway. A5 and HSG cells are derived from rat and human submandibular glands, respectively. Only A5 cells are derived from rat and human submandibular glands, respectively. Only A5 cells demonstrated such a pathway. The rate of A5 cell osmotic shrinkage was inhibited about fivefold in the presence of 300 m̈M HgCl2. To determine if this activity was associated with the expression of the prototypical water channel (aquaporin, AQP) AQP1, we used three separate experimental approaches; Northern analysis and reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of isolated mRNA, and Western analysis of cell membranes. All three methods yielded positive results with A5 cells and negative results with HSG cells. The ∼800 bp product of RT‐PCR was analyzed further by sequencing and restriction enzyme digestion. The results were consistent with the previously reported coding region sequence for rat kidney AQP1. The aggregate data demonstrate that marked differences in water‐permeability and water channel expression exist in these two salivary epithelial cell lines. © 1995 Wiley‐Liss, Inc.
Nucleolar p120 is expressed as a delayed early response gene and is inducible by DNA‐damaging agents
Authors: Fonagy Anna , Swiderski Carol , Freeman James W.
Published: February 2005

Regulation of the expression of the growth‐related nucleolar p120 protein was examined in serum‐deprived and stimulated nontransformed and SV40‐transformed WI‐38 human fibroblasts. In quiescent cells, transcriptional activity of the p120 gene was very low or undetectable, and the steady‐state levels of the p120 mRNA and the p120 protein were also negligible. The transient expression of the p120 gene in the cell cycle was detected in middle G1‐phase after the expression of the early response genes and before the expression of the DNA‐synthesis genes. Protein synthesis was required for the induction of p120 expression in serumstimulated cells. The increased level of p120 mRNA in middle G1‐phase was attributed to an increased transcription rate of the p120 gene, and not to a change in p120 mRNA stability. The calculated half‐life of p120 mRNA was unchanged (1.8 ± 0.2 hr) in all four cell conditions tested; i.e., in middle G1‐ or S‐phase cells and in exponentially growing normal or transformed cells. Transcription rate of the p120 gene was correlated with the steady‐state levels of either p120 protein or p120 mRNA. A sharp increase in p120 mRNA level occurred in both normal and transformed cells treated with actinomycin D used to examine p120 mRNA stability. This induction of p120 mRNA expression was seen in early G1‐phase, but not in quiescent cells, or in middle to late G1‐phase when cells expressed the highest level of p120 mRNA. The same expression pattern was seen by treatment with chlorambucil, another DNA‐damaging agent. The conclusions of these studies are that the expression of p120 (1) is serum inducible in a fashion characteristic of the delayed early response gene products, (2) requires the presence of newly synthesized proteins, (3) is regulated transcriptionally, and (4) can be induced by DNA‐damaging agents. © 1995 Wiley‐Liss, Inc.
Secretin potentiates cholecystokinin‐stimulated amylase release by AR4‐2J cells via a stimulation of phospholipase C
Authors: Bold Richard J. , Ishizuka Jin , Townsend Courtney M. , Thompson James C.
Published: February 2005

Alterations in the activity of phospholipase C (PLC) are thought to be the primary intracellular events leading to pancreatic acinar cell exocytosis of zymogen granules. When multiple hormones, each of which may stimulate different signal transduction pathways, bind to cell surface receptors, the cell must integrate these signals into a common response through communication (cross‐talk) among intracellular second messengers. We show that cholecystokinin (CCK) induces amylase secretion from AR4‐2J pancreatic acinar cells via stimulation of PLC activity. Secretin indirectly stimulated the PLC pathway through cross‐talk of the activated cAMP pathway to potentiate the CCK‐stimulated amylase secretion. Therefore, secretin potentiated the acinar cell secretory response to CCK by cAMP‐mediated cross‐talk with the PLC signal transduction pathway. © 1995 Wiley‐Liss Inc.
Changes in membrane potential during the progression of MCF‐7 human mammary tumor cells through the cell cycle
Authors: Wonderlin William F. , Woodfork Karen A. , Strobl Jeannine S.
Published: February 2005

We previously reported that MCF‐7 cells were arrested in the G0/G1 phase of the cell cycle by agents known to block the activity of ATP‐sensitive potassium channels (Woodfork et al., 1995, J. Cell Physiol. 162:163–171). The goal of our current study was to determine if MCF‐7 cells undergo changes in membrane potential during the cell cycle that might be linked to changes in K permeability. The resting membrane potentials of unsynchronized MCF‐7 cells during exponential growth phase were measured using sharp glass microelectrodes, and they ranged from −58.6 mV to −2.7 mV. The distribution of membrane potentials was best fitted by the sum of four Gaussian distributions with means of −9.0 mV, −17.4 mV, −24.6 mV, and −40.4 mV. These membrane potential groups were designated D (depolarized), ID (intermediate depolarized), IH (intermediate hyperpolarized), and H (hyperpolarized), respectively. The membrane potential was sensitive to the substitution of external K and Na but not Cl. The K:Na permeability ratio increased in proportion to the negativity of the membrane potential. MCF‐7 cells pharmacologically arrested in G0/G1 phase were depolarized compared to control, with cells shifted from the H and IH groups to the D group. Tamoxifen‐arrested cells chased from G0/G1 into S phase by the addition of mitogenic concentrations of 17β‐estradiol were not depolarized, and these cells were shifted from the D group back to the IH and H groups. We conclude that MCF‐7 cells hyperpolarize during passage through G0/G1 and into S phase, and this hyperpolarization probably results from an increase in the relative permeability of the plasma membrane to K. © 1995 Wiley‐Liss Inc.
Short circuiting stress protein expression via a tyrosine kinase inhibitor, herbimycin A
Authors: Hegde Ramanujan S. , Zuo Jianru , Voellmy Richard , Welch William J.
Published: February 2005

We set out to identify pharmacological means by which to activate the so‐called heat shock or stress response and thereby harness the protective effect afforded to the cell by its acquisition of a thermotolerant phenotype. An earlier report by Murakami et al. (1991, Exp. Cell Res., 195: 338–344) described the increased expression of the 70 kDa heat shock proteins in human A431 cells exposed to Herbimycin A (HA), a benzoquinoid ansamycin antibiotic. We show here that treatment of cells with HA results in the increased expression of all of the constitutively expressed stress proteins and confers upon the cells a thermotolerant‐like phenotype. Increases in the expression of the stress proteins continued for as long as the cells were exposed to the drug and was independent of the pre‐existing levels of the stress proteins. Unlike heat shock or other metabolic stressors, we did not observe any adverse cellular effects following HA exposure. For example, unlike most agents/treatments that elicit the stress response HA‐treated cells exhibited no obvious abnormalities with respect to protein maturation, protein insolubility, the integrity of the intermediate filament cytoskeleton, or overall cell viability. In addition, unlike other metabolic stressors, HA treatment did not result in the translocation of hsp 73 into the nucleus/nucleolus. Finally, for at least rodent cells, HA exposure did not result in any obvious activation of the heat shock transcription factor. Based on these findings, we suggest that HA treatment of cells results in a “short‐circuiting” of the pathway(s) that normally regulates the expression of the stress proteins. These results are discussed as they pertain to the potential use of HA in animals as a way to harness the protective effects afforded by the stress response. © 1995 Wiley‐Liss Inc.
Ouabain induces inhibition of the progression phase in human T‐cell proliferation
Authors: Brodie Chaya , Tordai Atilla , Saloga Joachim , Domenico Joanne , Gelfand Erwin W.
Published: February 2005

Ouabain, a specific inhibitor of the Na‐K ATPase, has been shown to exert immunosuppressive effects. The goals of this study were to define the stage of the proliferative response which is sensitive to ouabain and to correlate the inhibitory action of ouabain on cell proliferation with its effect on Na‐K ATPase activity. We found that ouabain inhibited T‐cell proliferation in a dose‐dependent manner and this inhibition was similar in CD4+ and CD8+ T cells. To define the role of the Na‐K ATPase in early activation of T lymphocytes, we examined the effects of ouabain on the induction of competence (acquisition of responsiveness to interleukin (IL)‐2 or IL‐4) by phytohemagglutinin (PHA) or the combination of phorbol dibutyrate/ionomycin. Ouabain, at concentrations that completely inhibited the enzyme activity, did not interfere with the induction of competence, suggesting that although activated cells express increased activity of Na‐K ATPase, this enzyme activity does not play a role in early activation pathways. In contrast, ouabain inhibited the progression phase to DNA synthesis in a dose‐dependent manner even at concentrations that had little or no effect on NaK ATPase activity. This inhibition was not due to a decrease in the production of IL‐2 but rather to an inhibition of the expression of the p55 and p75 subunits of the IL‐2 receptor (IL‐2R). The inhibition of p55 appeared to occur at the mRNA level. These results indicate that the activity of the NaK ATPase is not essential for the induction of competence or early activation. On the other hand, inhibition of cell proliferation and transcription of IL‐2R subunits by low concentrations of ouabain may be related to changes in intracellular K+ concentrations or to inhibition of membranal phospholipid metabolism secondary to alteration in NaK ATPase activity. © 1995 Wiley‐Liss, Inc.
Stem cell factor can overcome inhibition of highly purified human burst‐forming units‐erythroid by interferon γ
Authors: Dai Chun‐Hua , Krantz Sanford B. , Kollar Kathleen , Price James O.
Published: February 2005

Highly purified human blood burst‐forming units‐erythroid (BFU‐E) were used to study the effects of interferon γ (IFNγ). IFNγ inhibited erythroid colony formation, cell proliferation, and differentiation of day 3 to day 6 mature BFU‐E in a dose‐dependent manner. The primitive BFU‐E (day 1 and day 2 cells) and later day 7 cells were less affected. IFNγ dose‐response experiments demonstrated that the number and size of erythroid colonies were reduced at a concentration of 500 U/ml with more complete inhibition at 1,000 U/ml. Inhibition of day 4 to day 6 erythroid progenitors was first noted by 72 h of incubation with IFNγ, and target cell growth and differentiation continued to decrease with further incubation. IFNγ also induced erythroblast apoptosis which was demonstrated by both nuclear condensation and fragmentation plus flow cytometry with in situ end‐labelling. Because day 3 to day 6 cells need stem cell factor (SCF) for development in serum‐free culture, the relationship of IFNγ inhibition to this growth factor was investigated. The reduction in the number of erythroid colonies by IFNγ was reversed by SCF although the colony size was not completely re‐established. In contrast, interleukin‐3 did not have the capacity to overcome the inhibitory effects of IFNγ. Since IFNγ blood levels are elevated in some anemias of chronic disease, IFNγ may have a role in promoting this anemia and its inhibitory effect might be better overcome by SCF plus EP. However, the mechanism by which these growth factors overcome the inhibition of IFNγ, or vice versa, is unknown at the present time. © 1995 Wiley‐Liss, Inc.
Hepatocyte growth factor/scatter factor expressed in follicular papilla cells stimulates human hair growth in vitro
Authors: Shimaoka Shin , Tsuboi Ryoji , Jindo Toshimasa , Imai Ryusuke , Takamori Kenji , Rubin Jeffrey S. , Ogawa Hideoki
Published: February 2005

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional polypeptide which acts as mitogen, motogen, or morphogen. In this study, we examined the effect of HGF/SF on human hair growth using organ and cell culture systems. HGF/SF was found to stimulate hair length and DNA synthesis in hair follicles at increasing concentrations up to 10 ng/ml (P < 0.05 and P < 0.01, respectively). HGF/SF stimulated [3H]thymidine incorporation by hair bulb‐derived keratinocytes with the strongest response at 30 ng/ml of HGF/SF (P < 0.05). Cultured follicular papilla cells secreted HGF/SF, measured by an enzyme‐linked immunoassay, in response to interleukin 1‐α (IL1‐α, 10 ng/ml), tumor necrosis factor‐α (TNF‐α, 10 ng/ml), or tetradecanoylphorbolacetate (100 nM) at levels ranging from 0.2 to 0.3 ng/mg protein/48 h. HGF/SF mRNA expressions, measured by the reverse transcription‐polymerase chain reaction, were detected in follicular papilla cells, and were also stimulated by the three reagents. Transforming growth factor‐β (10 ng/ml) suppressed both protein and mRNA levels. These results suggest that hair follicle elongation induced by HGF/SF in organ culture occurs partly due to the mitogenic activity of HGF/SF expressed in follicular papilla cells on hair bulb‐derived keratinocytes. © 1995 Wiley‐Liss, Inc.
Activation of phosphotyrosine phosphatase activity is associated with decreased differentiation in adult bovine lens
Authors: Blanquet Pierre R. , Croquet Frédéric
Published: February 2005

The postnatal vertebrate eye lens provides an opportunity to study possible involvement of reversible protein phosphorylation in the differentiation process of epithelial cells. Epithelial cells at the lens equator, indeed, differentiate continuously into fiber cells throughout life but this capacity progressively decreases with age. Here we describe the characterization of a phosphotyrosine‐protein phosphatase(s) (PTPase(s)) in the equatorial epithelium of bovine lens which exhibits a high level of specific activity. PTPase(s) is detected in cellular detergent extracts using phospholabeled synthetic peptides, p‐nitrophenyl phosphate, and lens epithelial membranes as substrates. We show that activity of this PTPase(s) is increased in the equatorial epithelium as the age is increased. We also show that this enzyme(s) exerts its dephosphorylating activity predominantly on a calpactin‐like protein associated with lens epithelial membranes. Dephosphorylation of this protein is only obtained when membranes are subjected to extracts in the presence of fibroblast growth factor (FGF). It is suggested that an FGF‐activated PTPase(s) might conceivably counteract effects of differentiation stimulatory factors for limiting differentiation of lens throughout life. © 1995 Wiley‐Liss, Inc.
Second‐site mutations in cyclic AMP‐Sensitive revertants of a Ka mutant of S49 mouse lymphoma cells reduce the affinity of regulatory subunit of cyclic AMP‐Dependent protein kinase for catalytic subunit
Authors: Cauthron Robert D. , Gorman Karen B. , Symcox Marina M. , Steinberg Robert A.
Published: February 2005

Ka mutants of S49 mouse lymphoma cells are generally heterozygous for expression of wild‐type and mutant regulatory (R) subunits of type lα cyclic AMP‐(cAMP)‐dependent protein kinase, where the mutant R subunit has a defect in cAMP‐binding to one of two intrachain cAMP‐binding sites. Several cAMP‐sensitive revertants of such a Ka mutant were found previously to harbor second‐site mutations in the mutant allele, and we have now identified three such mutations by sequence analysis of PCR‐amplified cDNAs. The resulting amino acid changes were Ala98 to Thr, Gly179 to Arg, or Gly224 to Asp. The Ka mutation in these strains (Glu201 to Lys) eliminated cAMP‐binding to the more aminoterminal cAMP‐binding site (site A). None of the second‐site mutations restored this activity in bacterially expressed recombinant R subunit. On the other hand, all three second‐site mutations reduced the apparent affinity of the mutant R subunit for catalytic (C) subunit with the effects of the substitutions at Ala98 and Gly179 substantially greater than the effect of the substitution at Gly224. Patterns of phosphorylation and turnover of wild‐type and mutant R subunits in intact revertant cells were consistent with reduced association of the doubly mutant subunits with C subunit, but the free mutant subunits apparently were more stable than free wild‐type subunits. Differences in metabolic turnover of mutant and wild‐type subunits did not correlate with the sensitivities of the isolated proteins to proteolytic cleavage. © 1995 Wiley‐Liss, Inc.
Adenine nucleotide metabolism by chondrocytes in vitro: Role of ATP in chondrocyte maturation and matrix mineralization
Authors: Hatori Masashi , Teixeira Cristina C. , Debolt Kristine , Pacifici Maurizio , Shapiro Irving M.
Published: February 2005

The objective of the investigation was to explore the notion that chondrocytes in the growth plate secrete nucleotides and that these compounds are used to regulate cell maturation and matrix mineralization. Chondrocytes were isolated from the cephalic region of chick embryo sterna and maintained in culture until confluent. To promote expression of the mature phenotype, cultures were then treated with retinoic acid. During the culture period, medium was removed and analyzed for nucleotides using a modified reverse‐phase high‐performance liquid chromatography (HPLC) procedure. We found that culture medium, conditioned by the chondrocytes, contained significant quantities of nucleotides. Moreover, the nucleotide concentrations were similar in magnitude to levels reported for media conditioned by other cell types. In terms of species, adenosine diphosphate (ADP) was the major nucleotide present in the conditioned medium; adenosine monophosphate (AMP) was present, but at a lower concentration than ADP. To examine the possibility that adenosine triphosphate (ATP) was released by the cultured chondrocytes, but was rapidly degraded into ADP and AMP, we examined the kinetics of ATP breakdown by chondrocytes. We found that chondrocytes degraded over 70% of exogenous ATP within 15 minutes. Similar experiments performed with ADP and AMP indicated that these nucleotides were also degraded by the cells, but at a slower rate than ATP. To determine whether the extracellular nucleotides modulate cartilage development, we examined the effect of exogenous ATP on four major determinants of chondrocyte function: alkaline phosphatase activity, cell proliferation rate, anaerobic metabolism, and mineral deposition. We found that ATP caused only minimum alterations in cell number and alkaline phosphatase activity; however, it increased the lactate content of the medium probably by stimulating anaerobic glycolysis. We noted that ATP had a significant effect on the amount and type of mineral deposited into chondrocyte cultures. Compared with untreated controls, ATP stimulated formation of a small amount of poorly crystallized calcium phosphate. The results of the study show for the first time that chondrocytes release nucleotides into the extracellular milieu. Although they are rapidly degraded, they serve to regulate both mineral formation and energy metabolism. © 1995 Wiley‐Liss Inc.
Differential regulation of the expression of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase and pyruvate kinase by cyclic adenosine 3′,5′‐monophosphate in fetal and adult hepatocytes
Authors: Casado Marta , Boscá Lisardo , Martín‐Sanz Paloma
Published: February 2005

Incubation of fetal hepatocytes from 21‐day‐old rats with permeant derivatives of cyclic AMP (cAMP) or glucagon, increased the mRNA levels of 6‐phosphofructo‐2‐kinase/fructose 2,6‐bisphosphatase (PFK‐2/FBPase‐2), L‐pyruvate kinase (L‐PK) and phosphoenolpyruvate carboxykinase (PEPCK). Contrary to this behavior, adult hepatocytes exhibited a decrease in the PFK‐2/FBPase‐2 and L‐PK mRNA levels when incubated under equivalent experimental conditions. Dexamethasone also increased the PFK‐2/FBPase‐2 mRNA levels and costimulation of fetal hepatocytes with dexamethasone and a permeant analogue of cyclic AMP enhanced the levels of PFK‐2/FBPase‐2 mRNA, a situation opposite to that exhibited by adult hepatocytes. Treatment of hte hepatocytes with transcriptional and translational inhibitors also produced differential responses in both types of cells. The PFK‐2/FBPase‐2 mRNA in fetal hepatocytes was more stable than in the adult cells. These results suggest that specific transcriptional factors and regulatory pathways differentially operate in fetal and adult hepatocytes in the control of the responses of carbohydrate metabolism to cAMP. © 1995 Wiley‐Liss Inc.
Apoptosis: Programmed cell death in health and disease
Authors: John Bright J. , Khar Ashok
Published: February 2005

Apoptosis is a normal physiological cell death process of eliminating unwanted cells from living organisms during embryonic and adult development. Apoptotic cells are characterised by fragmentation of nuclear DNA and formation of apoptotic bodies. Genetic analysis revealed the involvement of many death and survival genes in apoptosis which are regulated by extracellular factors. There are multiple inducers and inhibitors of apoptosis which interact with target cell specific surface receptors and transduce the signal by second messengers to programme cell death. The regulation of apoptosis is elusive, but defective regulation leads to aetiology of various ailments. Understanding the molecular mechanism of apoptosis including death genes, death signals, surface receptors and signal pathways will provide new insights in developing strategies to regulate the cell survival/death. The current knowledge on the molecular events of apoptotic cell death and their significance in health and disease is reviewed.
Activation of mGluR5 modulates Ca2+currents in retinal amacrine cells from the chick
Authors: SOSA ROMINA , GLEASON EVANNA
Published: February 2005

In the inner plexiform layer, amacrine cells receive glutamatergic input from bipolar cells. Glutamate can depolarize amacrine cells by activation of ionotropic glutamate receptors or mediate potentially more diverse changesviaactivation of G protein-coupled metabotropic glutamate receptors (mGluR5). Here, we asked whether selective activation of metabotropic glutamate receptor 5 is linked to modulation of the voltage-gated Ca2+channels expressed by cultured GABAergic amacrine cells. To address this, we performed whole-cell voltage clamp experiments, primarily in the perforated-patch configuration. We found that agonists selective for mGluR5, including (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), enhanced the amplitude of the voltage-dependent Ca2+current. The voltage-dependent Ca2+current and CHPG-dependent current enhancement were blocked by nifedipine, indicating that L-type Ca2+channels, specifically, were being modulated. We have previously shown that activation of mGluR5 produces Ca2+elevations in cultured amacrine cells (Sosa et al., 2002). Loading the cells with 5 mM BAPTA inhibited the mGluR5-dependent enhancement, suggesting that the cytosolic Ca2+elevations are required for modulation of the current. Although activation of mGluR5 is typically linked to activation of protein kinase C, we found that direct activation of this kinase leads to inhibition of the Ca2+current, indicating that stimulation of this enzyme is not responsible for the mGluR5-dependent enhancement. Interestingly, direct stimulation of protein kinase A produced an enhancement of the Ca2+current similar to that observed with activation of mGluR5. Thus, activation of mGluR5 may modulate the L-type voltage-gated Ca2+current in these GABAergic amacrine cellsviaactivation of protein kinase A, possiblyviadirect activation of a Ca2+-dependent adenylate cyclase.
Retinal bipolar cells: Contrast encoding for sinusoidal modulation
and steps of luminance contrast
Authors: BURKHARDT DWIGHT A. , FAHEY PATRICK K. , SIKORA MICHAEL A.
Published: February 2005

Contrast encoding for sinusoidal modulations of luminance contrast
was investigated by intracellular recording in the intact salamander
retina. In what appears to be the first study of this kind for
vertebrate bipolar cells, responses of the central receptive-field
mechanism of cone-driven cells to modulation of 3 Hz were analyzed
quantitatively via both signal averaging and a Fast Fourier
Transform (FFT) while the retina was light adapted to 20
cd/m2. Depolarizing and hyperpolarizing bipolar cells
showed very similar encoding. Both responded with sinusoidal waveforms
whose amplitude varied linearly with modulation depths ranging up to
7–8%. The slope of the modulation/response curve was very
steep in this range. Thus, the contrast gain was high, reaching values
of 6–7, and the half-maximal response was achieved at modulations
of 9% or less. At modulations above ∼15%, the responses typically
showed strong compressive nonlinearity and the waveform was
increasingly distorted. At maximum modulation, the higher harmonics of
the FFT constituted about 30% of the amplitude of the fundamental.
Measurements were also made for cones and horizontal cells. Both cell
types showed predominantly linear responses and low contrast gain, in
marked contrast to bipolar cells. These results suggest that the high
contrast gain and strong nonlinearity of bipolar cells largely arise
postsynaptic to cone transmitter release. Further experiments were
performed to compare responses to contrast steps versus those to
sinusoidal modulation. In the linear range, we show that the contrast
gains of cones and horizontal cells are low and virtually identical for
both steps and sinusoidal modulations. In bipolar cells, on the other
hand, the contrast gain is about two times greater for steps than that
for the 3-Hz sine waves. These results suggest that mechanisms
intrinsic to bipolar cells act like a high-pass filter with a short
time constant to selectively emphasize contrast transients over slower
changes in contrast.
Local mechanisms for the separation of optic flow-field components in the land crab,Cardisoma guanhumi: A role for motion parallax?
Authors: JOHNSON AARON P. , BARNES W. JON. P. , MACAULEY MARTIN W.S.
Published: February 2005

Although a number of global mechanisms have been proposed over the years that explain how crabs might separate the rotational and translational components of their optic flow field, there has been no evidence to date that local mechanisms such as motion parallax are used in this separation. We describe here a study that takes advantage of a recently developed suite of computer-generated visual stimuli that creates a three-dimensional world surrounding the crab in which we can simulate translational and rotational optic flow. We show that, while motion parallax is not the only mechanism used in flow-field separation, it does play a role in the recognition of translational optic flow fields in that, under conditions of low overall light intensity and low contrast ratio when crabs find the distinction between rotation and translation harder, smaller eye movements occur in response to translation when motion parallax cues are present than when they are absent. Thus, motion parallax is one of many cues that crabs use to separate rotational and translational optic flow by showing compensatory eye movements to only the former.
Ocean oxygen isotope constraints on mechanisms for millennial‐scale climate variability
Authors: Olsen Steffen Malskær , Shaffer Gary , Bjerrum Christian J.
Published: February 2005

Millennial‐scale climate variability pervades the last several million years of Earth history with largest amplitudes during moderate glacial conditions. However, the processes behind such variability remain unclear. Here we present results from a simplified, coupled climate model that includes an explicit treatment of the atmosphere‐ocean cycle of oxygen‐18 isotope. The model exhibits a relatively strong North Atlantic overturning circulation for present day and last glacial maximum conditions. Self‐sustained, millennial‐scale oscillations with strong and weak overturning states are found for moderate glacial conditions. These oscillations reproduce observed features like “sawtooth” structure, Northern‐Southern Hemisphere asynchrony, sensitivity to climate state, and oxygen isotope signals in the ocean. Amplitudes, structures, and phasing of observed millennial‐scale variability in high‐resolution, benthic oxygen isotope records from the North Atlantic Ocean are consistent with large heating/cooling cycles in the ocean interior, as found in the model. Glacial meltwater, changes in solar irradiance and high‐frequency climate variability affect the timing, period, and persistence of the model climate cycles. However, the basic dynamics, structure, amplitude, and timescale of variability are due to internal, self‐sustained oscillations in the model ocean‐atmosphere system.
Oscillator clustering in a resource distribution chain
Authors: Postnov Dmitry E. , Sosnovtseva Olga V. , Mosekilde Erik
Published: February 2005

The paper investigates the special clustering phenomena that one can observe in systems of nonlinear oscillators that are coupled via a shared flow of primary resources (or a common power supply). This type of coupling, which appears to be quite frequent in nature, implies that one can no longer separate the inherent dynamics of the individual oscillator from the properties of the coupling network. Illustrated by examples from microbiological population dynamics, renal physiology, and electronic oscillator theory, we show how competition for primary resources in a resource distribution chain leads to a number of new generic phenomena, including partial synchronization, sliding of the synchronization region with the resource supply, and coupling-induced inhomogeneity.
Localization of a multiblock copolymer at a selective interface: Scaling predictions and Monte Carlo verification
Authors: Corsi Andrea , Milchev Andrey , Rostiashvili Vakhtang G. , Vilgis Thomas A.
Published: February 2005

We investigate the localization of a hydrophobic-polar regular copolymer at a selective solvent–solvent interface with emphasis on the impact of block length M on the copolymer behavior. The considerations are based on simple scaling arguments and use the mapping of the problem onto a homopolymer adsorption problem. The resulting scaling relations treat the gyration radius of the copolymer chain perpendicular and parallel to the interface in terms of chain length N and block size M, as well as the selectivity parameter χ. The scaling relations differ for the case of weak and strong localization. In the strong localization limit a scaling relation for the lateral diffusion coefficient D‖ is also derived. We implement a dynamic off-lattice Monte Carlo model to verify these scaling predictions. For chain lengths in a wide range (32⩽N⩽512) we find good agreement with the scaling predictions.
Cosurfactant and cosolvent effects on surfactant self-assembly in supercritical carbon dioxide
Authors: Chennamsetty Naresh , Bock Henry , Scanu Lauriane F. , Siperstein Flor R. , Gubbins Keith E.
Published: February 2005

The impact of alcohol additives on the self-assembly of surfactants in supercritical carbon dioxide is investigated using lattice Monte Carlo simulations. We observe that all studied (model) alcohols reduce the critical micelle concentration. The reduction is stronger the longer the hydrocarbon chain of the alcohol, and the higher the alcohol concentration. Short-chain alcohols are found to concentrate in the surfactant layer of the aggregates, replacing surfactant molecules and leading to a strong decrease of the aggregation number and a large increase of the number of aggregates. On the other hand, only a small number of alcohol molecules with longer chain length are found in the aggregates, leading to a slight increase in the aggregation number. However, structural properties such as size and density profiles of aggregates at the same aggregation number are not influenced markedly. Consequently, short-chain alcohols act as cosurfactants, directly influencing the properties of the aggregates, while alcohols with longer hydrocarbon chains work as cosolvents, altering the properties of the solvent. However, the transition between both extremes is gradual.
Revision of the second ionization energy of toluene
Authors: Roithová Jana , Schröder Detlef , Loos Jessica , Schwarz Helmut , Jankowiak Hans-Christian , Berger Robert , Thissen Roland , Dutuit Odile
Published: February 2005

Charge stripping (CS) of the molecular ion of toluene, C7H8+→C7H82++e, is often used as a reference for the determination of second ionization energies in energy-resolved CS experiments. For calibration of the kinetic energy scale, a value of IE(C7H8+)=(15.7±0.2)eV derived from the appearance energy of the toluene dication upon electron ionization has been accepted generally. Triggered by some recent discrepancies between CS measurements on the one hand and different experimental methods as well as theoretical predictions on the other, we have reinvestigated the photon-induced double ionization of toluene using synchrotron radiation. These photoionization measurements yield phenomenological appearance energies of AE(C7H8+)=(8.81±0.03)eV for the monocation and AE(C7H82+)=(23.81±0.06)eV for the dication. The former is in good agreement with a much more precise spectroscopic value, IE(C7H8)=(8.8276±0.0006)eV. Explicit consideration of the Franck–Condon envelopes associated with photoionization to the dication in conjunction with the application of the Wannier law leads to an adiabatic ionization energy IEa(C7H8+)=(14.8±0.1)eV, which is as much as 0.9eV lower than the previous value derived from electron ionization. Because in many previous CS measurements the transition C7H8+→C7H82++e was used as a reference, the energetics of several gaseous dications might need some readjustment.
Fractal dimensions of silica gels generated using reactive molecular dynamics simulations
Authors: Bhattacharya Sudin , Kieffer John
Published: February 2005

We have used molecular dynamics simulations based on a three-body potential with charge transfer to generate nanoporous silica aerogels. Care was taken to reproduce the sol-gel condensation reaction that forms the gel backbone as realistically as possible and to thereby produce credible gel structures. The self-similarity of aerogel structures was investigated by evaluating their fractal dimension from geometric correlations. For comparison, we have also generated porous silica glasses by rupturing dense silica and computed their fractal dimension. The fractal dimension of the porous silica structures was found to be process dependent. Finally, we have determined that the effect of supercritical drying on the fractal nature of condensed silica gels is not appreciable.
Evaluation of layer thickness in human teeth using higher-order-mode leaky Lamb wave interdigital transducers
Authors: Toda Shinji , Fujita Takeshi , Arakawa Hirohisa , Toda Kohji
Published: February 2005

An ultrasonic nondestructive evaluation technique of the layer thickness in human teeth is proposed using a leaky Lamb wave device with two arch-shaped interdigital transducers, operating at a plate∕water interface. The use of a higher-order-mode leaky Lamb wave with a phase velocity higher than the longitudinal wave velocity in the human tooth is essential to detect reflected ultrasound beams from the tooth section The layer thickness of dentin, estimated from the measured time interval between two reflected echoes, is in good agreement with the optically measured data.