bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of sperm in juvenile spermatogonial depletion (jsd) mutant mice is ascribed to a defect of intratubular environment to support germ cell differentiation
Authors: Mizunuma Masumi , Dohmae Kayoko , Tajima Youichi , Koshimizu Uichi , Watanabe Daisuke , Nishimune Yoshitake 
Published: February 2005

AbstractC57BL/6(B6)‐jsd/jsd mice are sterile due to the defective spermatogenesis in the testes. To know the cause of the deficient spermatogenesis in B6‐jsd/jsd mice, we examined whether the problem is within or outside the seminiferous tubules by transplanting tubules from cryptorchid testes of B6−+/+ mice into B6‐jsd/jsd testes or tubules from B6‐jsd/jsd mice into testes of (WB × C57BL/6)F1‐W/W (hereafter, WBB6F1‐W/W) mice. Type A spermatogonia differentiated into spermatids in seminiferous tubules from cryptorchid testes transplanted into B6‐jsd/jsd testes. In contrast, in B6‐jsd/jsd tubules transplanted into WBB6F1‐W/W testes, type A spermatogonia were stimulated to mitotic proliferation, but didn't proceed to any differentiated germ cells. The present results suggest that the cause of the deficient spermatogenesis in B6‐jsd/jsd mice is a defect of intratubular environment to support germ cell differentiation.
Regulation of NF‐κB activity in murine macrophages: Effect of bacterial lipopolysaccharide and phorbol ester
Authors: Vincenti Matthew P. , Burrell Terrie A. , Taffet Steven M. 
Published: February 2005

AbstractNuclear factor kappa‐B (NF‐κB) has been shown to play an important role in LPS‐mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP‐dependent protein kinase in the regulation of NF‐κB activity. In this study we have investigated the mechanism of NF‐κB induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF‐α NF‐κB element was transfected into the RAW264 macrophage‐like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8‐bromo cAMP. To further study NF‐κB induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF‐κB binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA‐binding activity confirmed these results and indicated that LPS induced a 55 KD DNA‐binding protein. Induction of this NF‐κB binding activity was not inhibited by pretreatment with the PKC inhibitor H‐7. H‐7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down‐regulates PKC, had no effect on LPS induction of NF‐κB activity in these cells. These results suggest that the induction of NF‐κB in macrophages by LPS is independent of PKC.
Identification of basic fibroblast growth factor sensitivity and receptor and ligand expression in human colon tumor cell lines
Authors: New Bonnie A. , Yeoman Lynn C. 
Published: February 2005

AbstractBasic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectoderrnal origin. Addition of exogenous bFCF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose‐response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth‐inhibitory effect of exogenous transforming growth factor‐beta (TGF‐β) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor‐specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF‐specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti‐bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.
Increased peptidylarginine deiminase expression during induction of prolactin biosynthesis in a growth‐hormone‐producing rat pituitary cell line, MtT/S
Authors: Nagata Saburo , Uehara Takemi , Inoue Kinji , Senshu Tatsuo 
Published: February 2005

AbstractInsulin and type I insulin‐like growth factor (IGF‐I) suppressed growth hormone (GH) expression followed by the induction of prolactin (PRL) biosynthesis in MtT/S cells cultured with normal sera. Insulin also increased the peptidylarginine deiminase activity in a dose‐dependent manner. The increase was detectable at 1 ng/ml and reached a maximum (about 16‐fold higher than the control) at 1 μg/ml. IGF‐I showed similar but less prominent effects. The enzyme activity started to increase by 15 hr after the addition of insulin (500 ng/ml), and reached a plateau level at 48 hr. There were concurrent increases in the enzyme mRNA level, enzyme biosynthesis, and enzyme protein contents detected by Northern blot hybridization, [35S]‐amino‐acid incorporation, and Western immunoblot analysis, respectively. Two‐color immunofluorescence staining at 1 day after the insulin addition detected a small number of peptidylarginine‐deiminase‐positive cells (about 1% of the total cells) which were also GH‐positive. The enzyme‐positive cells increased to 12% on day 2 and to 24–26% on days 4–6. PRL‐positive cells first appeared in the enzyme‐positive cell population on day 2, and PRL‐positive, enzyme‐negative cells appeared later. These results suggest that peptidylarginine deiminase expression increases in association with the hormone switching in MtT/S cells. When the cells were cultured in a steroid‐depleted medium, insulin failed to increase the enzyme activity. The insulin action could be specifically restored by estrogen, indicating estrogen‐insulin synergism in regulation of the enzyme expression.
Mapping the entry of reactive oxygen metabolites into target erythrocytes during neutrophil‐mediated antibody‐dependent cellular cytotoxicity
Authors: Petty Howard R. , Liang Bing , Maher Robert J. 
Published: February 2005

AbstractTransmitted Soret band optical microscopy has been used to image the entry and passage of reactive oxygen metabolites across target erythrocytes. Due to the rapid cytosolic diffusion of hemoglobin in comparison to video rates, it was necessary to use erythrocytes with relatively immobilized hemoglobin. To achieve this, erythrocytes from patients with sickle cell anemia were used. The movement of reactive oxygen metabolites across rabbit IgG‐opsonized sickle cells was observed in real time. These observations indicate that reactive oxygen metabolites can enter and cross targets in an asymmetric fashion.
Thrombin signal transduction mechanisms in human glomerular epithelial cells
Authors: He Ci‐Jiang , Peraldi M. N. , Adida C. , Rebibou J. M. , Meulders Q. , Sraer J. D. , Rondeau E. 
Published: February 2005

AbstractWe have previously shown that α‐thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase‐type (u‐PA) and tissue‐type plasminogen activator (t‐PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI‐1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose‐dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L‐type voltage‐sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin‐stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10−8 M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin‐stimulated synthesis of u‐PA, t‐PA, and PAI‐1. Both thrombin‐stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 μM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP‐inactivated thrombin which binds the thrombin receptor and γ‐thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u‐PA, t‐PA, and PAI‐1 synthesis although to a lesser extent than α‐thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by α‐thrombin in glomerular epithelial cells: (1) Ca2+ influx through a dihydroperidine‐sensitive calcium channel, which seems critical for mitogenesis; (2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u‐PA, t‐PA, and PAI‐1; (3) protein kinase C activation by other phospholipid breakdown can stimulate u‐PA, t‐PA, and PAI‐1 synthesis but not mitogenesis.
Common senescent cell‐specific antibody epitopes on fibronectin in species and cells of varied origin
Authors: Porter Mary Beth , Pereira‐Smith Olivia M. , Smith James R. 
Published: February 2005

AbstractThe phenomenon of in vitro cellular senescence has been demonstrated in cultured cells derived from humans and various other species. We have previously shown that monoclonal antibodies SEN‐1, SEN‐2, and SEN‐3 react to epitopes on fibronectin that are exposed when human diploid fibroblasts become senescent. We here present results demonstrating that exposure of these epitopes is specific to senescence for a variety of human cells: epidermal keratinocytes, mammary epithelial cells, as well as fibroblasts. Fibronectin from 11 additional species was also analyzed by Western immunoblot for ability to bind the SEN antibodies. SEN‐1 bound only human and gorilla fibronectin, whereas SEN‐2 and SEN‐3 bound fibronectin from those two species as well as the horse, cow, sheep, goat, dog, and chick. None of the antibodies reacted with fibronectin from the rabbit, rat, or mouse. These data indicated a correlation between the ability of the SEN antibodies to bind fibronectin from a particular species and the ability of cells from that species to exhibit a stable senescent phenotype in vitro. Therefore, exposure of this region of fibronectin may be important in the establishment and maintenance of cellular senescence. In addition, the ability of the SEN antibodies to react with fibronectin from a variety of senescent cells emphasizes their usefulness as markers for cellular senescence.
Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum‐Golgi complex
Authors: Mignatti Paolo , Morimoto Takashi , Rifkin Daniel B. 
Published: February 2005

AbstractBasic fibroblast growth factor (bFGF) modulates functions of a variety of cell types. Whereas bFGF is known to act extracellularly, the protein lacks a transient signal peptide. No defined mechanism for bFGF secretion has been characterized besides release from dead or injured cells. To study this problem we devised an experimental system to examine bFGF‐mediated migration of isolated single cells. Under these conditions individual cells are not affected by bFGF derived from other cells. By this method we have previously shown that bFGF released by NIH 3T3 cells transfected with bFGF cDNA modulates migration in an autocrine manner. We have now examined the effects on cell motility of drugs or treatments known to affect various pathways of protein secretion. Drugs that block secretion via the endoplasmic reticulum (ER)‐Golgi complex or via multidrug resistance proteins did not inhibit cell motility. Migration was enhanced by the calcium ionophore A23187, which stimulates exocytosis, and was inhibited by methylamine, serum‐free, and low temperature (18°C) conditions, which block endoand exocytosis. The reversal of these effects by the concomitant addition of affinity‐purified anti‐bFGF IgG or recombinant bFGF showed that the alterations in cell migration were mediated by changes in bFGF externalization. Thus bFGF can be released via a mechanism of exocytosis independent of the ER‐Golgi pathway. © 1992 Wiley‐Liss, Inc.
Differential extracellular matrix gene expression by fibroblasts during their proliferative life span in vitro and at senescence
Authors: Choi Augustine M. K. , Olsen David R. , Cook Keith G. , Deamond Scott F. , Uitto Jouni , Bruce Sarah A. 
Published: February 2005

AbstractIncreasing evidence supports the idea that the finite proliferative life span of normal fibroblasts is a differentiation‐like phenomenon. If this were correct, an ordered sequence of differential gene expression should be associated with the in vitro progression of cells from low passage to high passage (senescence). To define the pattern of expression of fibroblast differentiation‐associated genes during this in vitro progression, we have determined the temporal pattern of expression of extracellular matrix (ECM) genes in Syrian hamster dermal fibroblasts as a function of passage level and percentage of proliferative life span in vitro. Steady‐state mRNA levels were determined by Northern and dot blot analyses of total cellular RNA hybridized with cDNA probes specific for fibronectin, procollagen α1 III, and procollagen α1 I. Cells were analyzed at 24 hr postconfluence to minimize the presence of actively proliferating cells, and because maximal levels of fibronectin, α1 III, and α1 I mRNAs were observed 24 hr postconfluence. Unique, multiphasic patterns of expression of each of these ECM components were observed as the cells progressed from low passage to high passage. As the cells reached midhigh passage, fibronectin mRNA levels increased. This midpassage increase in fibronectin was followed by an increase in the level of α1 III mRNA as the cells reached the end of their in vitro proliferative life span, and then α1 I when the cells entered the postmitotic senescert phase, at which time the level of fibronectin mRNA also declined. A similar overlapping cascade pattern of up‐regulation of these genes is seen during development and wound repair. This suggests that as cultured fibroblasts reach the end of their proliferative life span, they reinitiate a gene expression program used in tissue development and repair. © 1992 Wiley‐Liss, Inc.
Inhibitors of protein synthesis cause increased hexose transport in cultured human fibroblasts by a mechanism other than transporter translocation
Authors: Germinario Ralph J. , Manuel Susannia , Chang Zully , Leckett Blaine 
Published: February 2005

AbstractWe have investigated the effect of various inhibitors of protein synthesis on hexose transport in human skin fibroblasts using 2‐deoxy‐D‐glucose (2‐DG) and 3‐0‐methyl‐D‐glucose (3‐OMG) to measure hexose transport. Exposure of glucose‐fed, serum‐free cultures to cycloheximide (CHX) (50 μg/ml) for 6 h resulted in increased 2‐DG transport (3.81 ± .53 vs. 6.62 ± .88 nmoles/mg protein/2 min; n = 9) and 3‐OMG transport (1.36 ±.66 vs. 3.18 ± .83 nmoles/mg protein/30 sec; n = 4) in the CHX exposed group. Under these conditions inhibition of protein synthesis was greater than 90%. This CHX induced transport increase was time dependent (approaching maximum within 1 h of exposure to CHX) and related to an increase in the Vmax of hexose transport in the CHX exposed group (18.4 ± 2.4 vs. 4.8 ± 1.1 nmoles 2‐DG/mg protein/min) with no difference in the transport Km (1.55 ± .63 vs. 2.92 ± .59 mM). Further, the CHX induced increase in hexose transport was reversible. Exposure of human fibroblasts to inhibitors of protein synthesis with different mechanisms of action (e.g., puromycin, pactamy‐cin, or CHX) all generated hexose transport increases in a concentration‐dependent fashion correlating with their increasing inhibitory effects on protein synthesis. Nucleotidase enriched (i.e., plasma membrane) fractions of control and CHX‐exposed cells showed no differences in D‐glucose inhibitable cytochalasin B binding activity. Further, quantitative Western analysis of nucleotidase enriched fractions indicated CHX exposure resulted in no significant increase in glucose transporter mass compared with control plasma membrane fractions. Glucose deprived cells, however, which exhibited increased sugar transport comparable to the CHX‐exposed group, did show increased glucose transporter mass in the plasma membrane fraction. The data indicate that inhibitors of protein synthesis can cause a significant elevation in hexose transport and that the hexose transporter mass in the isolated plasma membrane fractions did not reflect the whole cell transport change. It is suggested that a mechanism other than glucose transporter translocation to the plasma membrane may be involved in causing this sugar transport increase. © 1992 Wiley‐Liss, Inc.
Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury
Authors: Bhat Ganesh B. , Tinsley Steven B. , Tolson J. Keith , Patel Jawaharlal M. , Block Edward R. 
Published: February 2005

AbstractThe effect of hypoxia on subsequent susceptibility of porcine pulmonary artery endothelial cells (PAEC) to hydrogen peroxide (H2O2) injury was studied. Preexposure of PAEC to hypoxia for 3 or more h significantly increased susceptibility to subsequent H2O2 challenge. Analysis of the activities of antioxidant enzymes and xanthine oxidase/dehydrogenase suggested that changes in these enzymes in hypoxic PAEC were not responsible for the increased susceptibility. However, hypoxia resulted in significant time‐dependent decreases in total glutathione at 12 h or more. The rate of glutathione regeneration in diethylmaleate‐treated PAEC and the rate of uptake of cystine and glycine were significantly lower during hypoxia. Hypoxia also caused depletion of ATP and NADPH levels in PAEC, but these did not occur until well after hypoxia‐enhanced susceptibility to H2O2 injury was demonstrable. Alterations in glutathione levels and enhanced susceptibility were reversible when hypoxic PAEC were returned to normoxia. These results indicate that hypoxia increased the susceptibility to H2O2 injury by decreasing the ability of PAEC to maintain and regenerate cellular glutathione content in response to H2O2 challenge. © 1992 Wiley‐Liss, Inc.
Plasmin generation induces neutrophil aggregation: Dependence on the catalytic and lysine binding sites
Authors: Ryan Thomas J. , Lai Linda , Malik Asrar B. 
Published: February 2005

AbstractWe established that plasmin (10−10M to 10−6M) caused neutrophils (PMN) to aggregate using an in vitro assay. Plasminogen had no PMN aggregatory activity even at a concentration of 2 μM. However, plasminogen caused PMN to aggregate when incubated with plasminogen activators [tissue plasminogen activator (25–200 U/ml) or urokinase (5–500 U/ml)]. Tissue plasminogen activator and urokinase alone had no PMN aggregatory activity. Analysis of these incubation mixtures indicated that plasmin was generated in the process and that the time course of plasmin generation correlated with the aggregation response. Active‐site‐inhibited plasmin did not induce PMN aggregation, indicating that a functional catalytic site was required for the response. Pretreatment of PMN with either active‐site‐inhibited plasmin or tranexamic acid prevented PMN aggregation by plasmin, indicating that both binding of plasmin to the cell surface via the lysine binding sites and catalysis were required for the response. The generation of plasmin during activation of fibrinolysis may play a pro‐inflammatory role by mediating aggregation of PMN. © 1992 Wiley‐Liss, Inc.
Partial purification and characterisation of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy
Authors: Woodman Anthony C. , Selden Clare A. , Hodgson Humphrey J. F. 
Published: February 2005

AbstractWe have investigated the influences that nonparenchymal cells from regenerating rat liver exert on hepatocyte proliferation. When primary adult rat hepatocytes isolated from resting liver were co‐cultured with nonparenchymal cells (NPCs) from resting liver of a different syngeneic animal, the proliferative response of hepatocytes to epidermal growth factor (EGF) was unaffected by the presence of NPCs. In the presence of NPCs taken from livers that had undergone partial hepatectomy 24 hours before (regen‐NPCs), the response of hepatocytes from resting liver to EGF, TGF‐α, and hepatocyte growth factor (HGF) was markedly inhibited. Inhibitory activity was not dependent on cell‐to‐cell contact, and conditioned‐medium from regen‐NPCs, but not normal NPCs, inhibited EGF‐induced hepatocyte DNA synthesis by approximately 50%. After concentration by gel chromatography and lyophilisation, inhibition was 98%. The inhibitory activity migrated on SDS‐PAGE gel electrophoresis with an apparent molecular weight of 14 to 17 kDa and was trypsin‐sensitive but relatively heat‐stable. The effects of blocking antibodies established that it was not TGF‐β1, IL 1‐β, or IL 6. Investigations of regen‐NPCs taken at different time points demonstrated that inhibitory activity was released into conditioned medium of cells harvested at 24 and 48 hours after partial hepatectomy, but not 10 or 72 hours. This powerful inhibitor of hepatocyte response to proliferogens is released by cultures of NPCs with a time course suggesting that it may be involved in terminating the surge of hepatocyte replication induced by partial hepatectomy. © 1992 Wiley‐Liss, Inc.