bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractTaurine (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

AbstractThe 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

AbstractThe 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

AbstractVarious 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

AbstractWe 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

AbstractPrimary 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

AbstractRetinoic 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

AbstractThe 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

AbstractThe 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

AbstractThrombin, 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

AbstractThe 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

AbstractThe 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.