bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody to the phosphomannosyl receptor inhibits recycling of receptor in fibroblasts
Authors: Nolan Catherine M. , Creek Kim E. , Grubb Jeffrey H. , Sly William S.
Published: December 2004

The 215‐kd phosphomannosyl receptor is involved in the transport of newly synthesized acid hydrolases to lysosomes and also mediates the pinocytosis of lysosomal enzymes by fibroblasts in culture. Recycling of receptors to the sorting sites is an integral part of both these processes. In this report, we describe the inhibition in human fibroblasts of both functions of the phosphomannosyl receptor by a rabbit. Antiserum to the bovine liver receptor. This inhibition cannot be completely accounted for by inhibition of ligand‐receptor interaction. Rather the antibody appears to cross‐link receptors and cause a removal of receptors from the sorting sites (plasma membrane and Golgi apparatus) and their accumulation in a compartment from which they do not recycle. Removal of receptors from the recycling pool by antibody is irreversible, and return of receptors requires synthesis of new protein. Degradation of “trapped receptors” is enhanced (t½ = 7.5 hr), but much more gradual than their removal from the functional receptor pool (t:½ = 30min).
2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin receptors regulate transcription of the cytochrome P1‐450 gene
Authors: Durrin Linda K. , Jones Phillip B. C. , Fisher Joan M. , Galeazzi Donna R. , Whitlock James P.
Published: December 2004

The environmental contaminant 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) dioxin, produces a diverse set of biological responses which, in some cases, reflects the altered expression of specific genes. An intracellular receptor protein binds TCDD saturably and with high affinity and mediates several of TCDD's biological effects. In mouse hepatoma cells, TCDD induces aryl hydrocarbon hydroxylase activity by activating the transcription of the cytochrome P1‐450 gene. Studies of receptor‐defective variant cells indicate that the activation of cytochrome P1‐450 gene transcription requires functional TCDD receptors. Analysis of the DNA that flanks the 5′‐end of the mouse cytochrome P1‐450 gene reveals at least three control regions: a promoter, an inhibitory element, and a dioxin‐responsive element (DRE). Therefore, expression of the cytochrome P1‐450 gene represents a balance between negative and positive control. The DRE contains two discrete, non‐overlapping DNA domains that respond to TCDD. Each TCDD‐responsive domain acts independently of the other, each requires TCDD receptors for function, and each has the properties of a transcriptional enhancer. For example, the function of the DREs is relatively independent of both their location and their orientation with respect to the promoter. Together, the DREs and the TCDD‐receptor complex constitute a dioxin‐responsive enhancer system. Exposure of cells to TCDD results in the protection of a specific DNA domain from exonuclease digestion. This protection requires TCDD receptors. The protected domain maps to a DRE. This observation implies that the TCDD‐receptor complex interacts with the DRE to activate the transcription of the cytochrome P1‐450 gene.
Genetic evidence that the steroid‐regulated trafficking of cell surface glycoproteins in rat hepatoma cells is mediated by glucocorticoid‐inducible cellular components
Authors: Firestone Gary L. , John Nancy J. , Haffar Omar K. , Cook Paul W.
Published: December 2004

The biological control of posttranslational maturation and compartmentalization reactions that operate upon proteins during transport to their final cellular desti‐ nations is crucial for normal cellular function. Using the expression of mouse mammary tumor virus (MMTV) glycoproteins as sensitive probes in the viral‐ infected rat hepatoma cell line M1.54, we have discovered and documented a novel glucocorticoid‐regulated trafficking pathway that controls the cell surface localization of MMTV glycoproteins. One complement‐selected derivative of M1.54 cells, CR4, failed to compartmentalize cell surface MMTV glycoproteins in the presence of dexamethasone. To test genetically if this glycoprotein trafficking pathway is mediated by cellular or viral gene products, CR4 cells were fused with uninfected Fu5 rat hepatoma cells. Indirect immunofluorescence of CR4 × Fu5 heterokaryons revealed that Fu5 complemented the defect in CR4 only after exposure to 1 μM dexamethasone. The glucocorticoid inhibition of Fu5 proliferation was exploited to recover the receptor‐deficient uninfected derivative EDR3 that expressed a 100‐fold lower level of [3H]dexamethasone binding activity. Analysis of CR4 × EDR3 cell fusions by indirect immunofluorescence revealed that EDR3 cells complemented CR4 in a dexamethasone‐dependent manner, suggesting that EDR3 supplied a functional trafficking component while CR4 provided a functional glucocorticoid receptor to the heterokaryons. Taken together, our results demonstrate that cellular‐encoded glucocorticoid‐inducible components mediate the regulated trafficking of cell surface MMTV glycoproteins.
Heterogeneity of fibroblast response in host‐tumor cell‐cell interactions in metastatic tumors
Authors: Dabbous Mustafa Kh. , Haney Lena , Carter Lee M. , Paul A. K. , Reger James
Published: December 2004

The spread and invasion of tumor cells into host tissues are associated with the release of elevated levels of collagenolytic activity of both host and tumor cell origins. However, the mechanisms of regulation of the enzyme activity is still unresolved. Histological examination of human and animal tumors revealed morphological changes in stromal fibroblasts and mast cells at the tumor periphery. Numerous mast cells appeared at microfoci along the tumor: host tissue junction and mast cell degranulation were associated with collagenolysis. In vitro studies, using rat mammary adenocarcinoma and human lung adenocarcinoma cells, showed that both tumor cells and host fibroblasts participate in matrix degradation. Tumor‐associated stromal fibroblasts released higher levels of enzyme activity than normal fibroblasts and were more responsive to stimulation by tumor‐conditioned media and soluble mast cell products. Host fibroblasts appear to be heterogeneous populations of responsive and nonresponsive subpopulations based on their response to tumor‐ or mast‐cell‐mediated stimulation of collagenase release. Fibroblast subpopulations were obtained by density fractionation of serum‐deprived, synchronized confluent fibroblasts on discontinuous Percoll gradient. Density‐fractionated fibroblast subpopulations differed in their response to stimulation by mast cell products and tumor‐cell‐conditioned media. The stimulatory activity of tumor‐cell‐conditioned media also varied as a function of the metastatic potential of the tumor cells. The data suggest that cellular interactions between tumor cells and select subpopulations of host fibroblasts at the tumor periphery play a key role in host tissue degradation. However, heterogeneity of stromal fibroblasts may determine the site and extent of the tissue damage at foci of tumor invasion.
Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis
Authors: Muramatsu Takashi
Published: December 2004

Cell surface carbohydrates undergo marked alterations during mouse embryogenesis. In preimplantation embryos, many carbohydrate markers show stage‐specific expression in diverse ways. In early postimplantation embryos, certain carbohydrate markers are localized in defined regions in the embryo. Important carriers of stage‐specific carbohydrates are the lactoseries structure (Galβ1→4GlcNAc) and the globoseries structure (Galα1→4Gal). Notably, the glycoprotein‐bound large carbohydrate of poly‐N‐acetyllactosamine‐type ([Galβ1→4GlcNAcβ1 → 3]n) carries a number of markers preferentially expressed in early embryonic cells. These markers are of practical value in analyzing embryogenesis and cell differentiation. For example, in order to monitor in vitro differentiation of multipotential embryonal carcinoma cells, stage‐specific embryonic antigen‐1 (SSEA‐1) and the Lotus agglutinin receptor have been used as markers of the undifferentiated cells, and the Dolichos agglutinin receptor has been used as a marker of extraembryonic endoderm cells. Developmental control of cell surface carbohydrates is attained by controlled expression of activities of key glycosyltransferases; for example, the activity of N‐acetylglucosaminide αl → 3 fucosyltransferase is lost during in vitro differentiation of embryonal carcinoma cells to parietal endoderm cells, in parallel to the disappearance of SSEA‐1. Accumulating evidence suggests that poly‐N‐acetyllactosamine‐type glycans that are abundant in early embryonic cells are involved in cell surface recognition of these cells.
Inhibition of endocytosis from coated pits by acidification of the cytosol
Authors: Sandvig Kirsten , Olsnes Sjur , Petersen Ole W. , van Deurs Bo
Published: December 2004

Binding and endocytosis of the ligands transferrin, epidermal growth factor (EGF), and ricin were measured in a number of different cell lines after treatment of cells with compounds that react with SH‐groups and under conditions where the cytosolic pH was lowered. N‐ethylmalemide and diamide irreversibly inhibited endocytosis of all ligands tested, whereas low pH in the cytosol strongly inhibited endocytosis of transferrin and EGF. Data obtained by electron microscopy indicated that the formation of coated vesicles from coated pits is inhibited in acidified cells. Entry of ricin was much less affected, and ricin endocytosed under these conditions was able to intoxicate the cells. At low pH in the cytosol there was a calcium‐dependent increase in the number of transferrin receptors at the cell surface. The increase was even larger in the presence of the calcium ionophore A23187, whereas it was completely blocked by the calmodulin antagonists trifluoperazine and W7. The results show that endocytosis from coated pits can be inhibited in a reversible way by acidification of the cytosol and they suggest that a second pathway of endocytosis exists, possibly involving formation of vesicles from uncoated areas of the membrane.
ERV3 Human endogenous provirus mRNAs are expressed in normal and malignant tissues and cells, but not in choriocarcinoma tumor cells
Authors: Cohen Maurice , Kato Nobuyuki , Larsson Erik
Published: December 2004

Messenger RNA expression of a human endogenous provirus, ERV3, has been characterized in 170 specimens of normal and malignant human tissues and cells. In contrast to the high expression in first‐trimester and full‐term placental chorionic villi, most other human tissues expressed ERV3 mRNAs at a level of 2–30% of placenta. However, ERV3 mRNAs were not detected in choriocarcinoma tumor cell lines. These studies suggest that the ERV3 provirus may have been preempted for a biological function and disruption of its mRNA expression results in choriocarcinoma.
Heparanases and tumor metastasis
Authors: Nakajima Motowo , Irimura Tatsuro , Nicolson Garth L.
Published: December 2004

The successful penetration of endothelial basement membranes is an important process in the formation of hematogenous tumor metastases. Heparan sulfate (HS) proteoglycan is a major constituent of endothelial basement membranes, and we have found that HS‐degradative activities of metastatic B16 melanoma sublines correlate with their lung‐colonizing potentials. The melanoma HS‐degrading enzyme is a unique endo‐β‐D‐glucuronidase (heparanase) that cleaves HS at specific intrachain sites and is detectable in a variety of cultured human malignant melanomas. The treatment of B16 melanoma cells with heparanase inhibitors that have few other biological activities, such as N‐acetylated N‐desulfated heparin, results in significant reductions in the numbers of experimental lung metastases in syngeneic mice, indicating that heparanase plays an important role in melanoma metastasis. HS‐degrading endoglycosidases are not tumor‐specific and have been found in several normal tissues and cells. There are at least three types of endo‐β‐D‐glucuronidases based on their substrate specificities. Melanoma heparanase, an Mr ∼96,000 enzyme with specificity for β‐D‐glucuronosyl‐N‐acetylglucosaminyl linkages in HS, is different from platelet and mastocytoma endoglucuronidases. Elevated levels of heparanase have been detected in sera from metastatic tumor‐bearing animals and malignant melanoma patients, and a correlation exists between serum heparanase activity and extent of metastases. The results suggest that heparanase is potentially a useful marker for tumor metastasis.
Assessment of biological activity of synthetic fragments of transforming growth factor‐alpha
Authors: Darlak Krzysztof , Franklin Glen , Woost Philip , Sonnenfeld Elaine , Twardzik Daniel , Spatola Arno , Schultz Gregory
Published: December 2004

Transforming growth factor‐alpha (TGF‐α) is a single chain polypeptide hormone of 50 amino acids that stimulates growth of some human cancer cells via an autocrine mechanism. The domain(s) of TGF‐α that bind and activate its receptor have not been reported. Hydrophilicity plots of TGF‐α indicate three discrete sequences that are theoretically exposed on the hormone's surface and thus potentially able to interact with the TGF‐α receptor. Fragments of TGF‐α encompassing these hydrophilic domains were prepared by using solid‐phase peptide synthesis (SPPS) techniques and purified by use of high performance liquid chromotography (HPLC). Assessment of biological activity of the TGF‐α fragments indicated that none of the fragments significantly inhibited binding of EOF to the receptor, stimulated DNA synthesis of cells, inhibited EGF‐induced DNA synthesis of cells, stimualted growth of cells in soft agar, or induced phosphorylation of the receptor or p35 protein. These results indicate that the receptor binding domain of TGF‐α is not totally encompassed by any of the separate fragments tested and probably is formed by multiple separate regions of TGF‐α.
Human cytokines, tumor necrosis factor, and interferons: Gene cloning, animal studies, and clinical trials
Authors: Bollon Arthur P. , Berent Susan L. , Torczynski Richard M. , Hill Norwood O. , Lemeshev Yuri , Hill Joseph M. , Jia Feng Lan , Joher Anwar , Pichyangkul Sathit , Khan Amanullah
Published: December 2004

Presented is a comprehensive program designed to isolate human cytokine genes and investigate their relative induction, and to analyze cytokine activities in cell culture, animal tumor models, and human clinical trials. Human cytokine cDNAs have been isolated from a cDNA library made from normal human peripheral blood leukocytes (PBLs) treated with Sendai virus and the relative induction of tumor necrosis factor (TNF), alpha and gamma interferons (IFN‐α, IFN‐γ), and interleukin‐1 beta IL‐1β genes has been analyzed. In the Sendai virus‐induced PBL system, IL‐1β mRNA was shown to be approximately twofold higher than TNF or IFN‐α mRNA whereas IFN‐γ mRNA was 50–100‐fold lower than TNF or IFN‐α mRNA. The cytotoxic activity of TNF was analyzed on several cell lines and IFN‐α and IFN‐γ were shown to potentiate TNF cytotoxicity about 2–200‐fold depending on cell lines. The LD50 for recombinant TNF in BALB/c mice was determined to be 6 × 107 U/kg and the therapeutic dose of recombinant TNF in sarcoma 180 bearing BALB/c mice was 3 × 105 U/kg, indicating a wide therapeutic index. Phase I clinical trials of recombinant TNF given I.V. indicated a tolerated dose of 150,000 U/kg with biphasic half‐life (T‐1/2) of 2 and 31 min following TNF injection. Phase II trials of TNF and trials of TNF combined with IFN‐α are in progress. These studies indicate that cytokines such as TNF and IFN‐α are subject to similar induction systems, potentiate each other's activities, and can be tolerated at specific doses for potential therapeutic use.
Calmodulin plays a dominant role in determining neurotransmitter regulation of neuronal adenylate cyclase
Authors: Cooper Dermot M. F. , Ahlijanian Michael K. , Perez‐Reyes Edward
Published: December 2004

Ca2+, through the mediation of calmodulin, stimulates the activity of brain adenylate cyclase. The growing awareness that fluctuating Ca2+, concentrations play a major role in intracellular signalling prompted the present study, which aimed to investigate the implications for neurotransmitter (receptor) regulation of enzymatic activity of this calmodulin regulation. The role of Ca2+/calmodulin in regulating neurotransmitter‐mediated inhibition and stimulation was assessed in a number of rat brain areas. Ca2+/calmodulin stimulated adenylate cyclase activity in EGTA‐washed plasma preparations from each region studied—from 1.3‐fold (in striatum) to 3.4‐fold (in cerebral cortex). The fold‐stimulation produced by Ca2+/calmodulin was decreased in the presence of GTP, forskolin, or Mn2+. In EGTA‐washed membranes, receptor‐mediated inhibition of adenylate cyclase was strictly dependent upon Ca2+/calmodulin stimulation in all regions, except striatum. A requirement for Mg2+ in combination with Ca2+/calmodulin to observe neurotransmitter‐mediated inhibition was also observed. In contrast, receptor‐mediated stimulation of activity was much greater in the absence of Ca2+/calmodulin. The findings demonstrate that ambient Ca2+ concentrations, in concert with endogenous calmodulin, may play a central role in dictating whether inhibition or stimulation of adenylate cyclase by neurotransmitters may proceed.
Detection of GD3 ganglioside in childhood acute lymphoblastic leukemia with monoclonal antibody to GD3: Restriction to immunophenotypically defined T‐cell disease
Authors: Merritt William D. , Sztein Marcelo B. , Reaman Gregory H.
Published: December 2004

We have recently reported that the disialoganglioside GD3 is found in cellular lipid extracts of T‐cell acute lymphoblastic malignancies (T‐ALL) but is not detectable by resorcinol staining in extracts of non‐T acute lymphoblastic leukemia blasts (non‐T‐ALL). We have now extended this study to assess the detectability of GD3 in T‐ALL vs non‐T‐ALL utilizing an anti‐GD3 antibody, R24. Gangliosides isolated from T‐ALL and non‐T‐ALL blasts by two different methods were separated by thin‐layer chromatography and stained with anti‐GD3 and a control antibody specific for GM3 and sialosylparagloboside (SPG). Anti‐GD3 reactivity was observed in extracts from T‐ALL cells in all cases, whereas GD3 was not detected in any of the non‐T‐ALL samples. The anti‐GM3/SPG antibody stained GM3 in all of the leukemic samples analyzed as well as SPG in the non‐T‐ALL samples. Indirect immunofluorescence was used to assess the expression of GD3 at the surface of leukemic blasts. Fluorescence‐activated cell sorting analysis with R24 showed that whereas T‐ALL blasts were highly reactive with this antibody, non‐T‐ALL blasts were totally unreactive. In an analysis of a larger number of leukemia patients by fluorescence microscopy, 20 out of 28 samples with the T‐ALL phenotype were positive for R24, whereas zero out of 11 non‐T‐ALL samples were reactive. These results confirm our earlier finding of the specificity of GD3 to the T‐ALL subclass of childhood leukemias and furthermore suggest the potential value of anti‐GD3 as an immunological tool for the diagnosis and therapy of T‐cell ALL.
Structures, function, and transformational changes of the sugar chains of glycohormones
Authors: Kobata Akira
Published: December 2004

Human chorionic gonadotropin (hCG), human luteinizing hormone, human thyroid‐stimulating hormone, and human follicle‐stimulating hormone are closely related family of proteins which share a common α‐subunit. However, their sugar moieties are quite different.

hCG contains five acidic asparagine‐linked sugar chains. These five sugar chains are derived by sialylation from three neutral oligosaccharides: two biantennary (N‐1 and N‐2) and one monoantennary (N‐3) complex‐type oligosaccharides. Although hCG purified from the urine of pregnant women is more enriched in sialylated sugar chains than that purified from placenta, the molar ratio of N‐1, N‐2, and N‐3 of these two hCGs are the same (1:2:1). Comparative study of the sugar moieties of the α‐ and β‐subunits of hCG revealed that α contains 1 mol each of N‐2 and N‐3, while β contains 1 mol each of N‐l and N‐2. This specific distribution of oligosaccharides at the four asparagine loci of the hCG molecule is now helping us to consider the functional role of the sugar moiety of glycohormones.

hCG is produced not only by the trophoblast but also by various trophoblastic diseases. The hCGs purified from the urine of patients with hydatidiform mole contain the same oligosaccharides as normal hCG. However, those from the urine of choriocareinoma patients contain five additional neutral oligosaccharidcs. In contrast, hCGs from invasive‐mole patients contain three of the live oligosaccharidcs, specifically found in choriocarcinoma hCGs.

The malignant transformational change of the sugar moiety of hCG can be explained by an increase of a fucosyltransferase, which forms the Fucαl→6GlcNAc group and by ectopic expression and subsequent modification of N‐acetylglucosaminyltransferase IV. The appearance of tumor‐specific sugar chains of hCG has been used to develop a new diagnostic method for invasive mole and choriocarcinoma.
Lymphocyte activation and capping of hormone receptors
Authors: Bourguignon Lilly Y. W. , Jy Wenche , Majercik Mary H. , Bourguignon Gerard J.
Published: December 2004

In this study both a Ligand‐dependent treatment [concanavalin A (Con A)] and a ligand‐independent treatment [high‐voltage pulsed galvanic stimulation (HVPGS)] have been used to initiate lymphocyte activation via a transmembrane signaling process. Our results show that both treatments cause the exposure of two different hormone [insulin and interIeukin‐2 (IL‐2)] receptors within the first 5 min of stimulation. When either insulin or IL‐2 is present in the culture medium, the stimulated lymphocytes undergo the following responses: (1) increased free intra‐cellular Ca2+activity; (2) aggregation of insulin or IL‐2 receptors into patch/cap structures; (3) tyrosine‐kinase‐specific phosphorylation of a 32‐kd membrane protein; and finally (4) induction of DNA synthesis.

Further analysis indicates that hormone receptor capping is inhibited by (1) cytochalasin D, suggesting the involvement of microfilaments; (2) sodium azide, indicating a requirement for ATP production; and (3) W‐5, W‐7, and W‐12 drugs, implying a need for Ca2 +/calmodulin activity. Treatment with these metabolic or cytoskeletal inhibitors also prevents both the tyrosine‐kinase‐specific protein phosphorylation and DNA synthesis which normally follow hormone receptor capping. Double immunofluorescence staining shows that actomyosin, Ca2+/calmodulin, and myosin light‐chain kinase are all closely associated with the insulin and IL‐2 receptor cap structures.

These findings strongly suggest that an actomyosin‐mediated contractile system (regulated by Ca2+, calmodulin, and myosin light‐chain kinase in an energy‐dependent manner) is required not only for the collection of insulin and IL‐2 receptors into patch and cap structures but also for the subsequent activation of tyrosine kinase and the initiation of DNA synthesis. We, therefore, propose that the exposure and subsequent patching/capping of at least one hormone receptor are required for the activation of mouse splenic T‐Iymphocytes.
Vanadate‐treated baby hamster kidney fibroblasts show cytoskeleton and adhesion patterns similar to their rous sarcoma virus‐transformed counterparts
Authors: Marchisio Pier Carlo , D'Urso Nicoletta , Comoglio Paolo M. , Giancotti Filippo G. , Tarone Guido
Published: December 2004

Rous sarcoma virus‐trans formed baby hamster kidney fibroblasts (RSV/B4‐BHK) adhere to a fibronectin‐coated substratum by means of dot‐like adhesion sites called podosomes in view of their shape and function as cellular feet (Tarone et al.: Exp Cell Res 159:141, 1985). Podosomes concentrate tyrosine‐phosphorylated proteins, including pp60v‐src, and appear in many cells transformed by oncogenes coding for tyrosine kinases. In this paper we used orthovanadate, an inhibitor of phosphotyrosine phosphatases, in order to increase the cellular concentration of phosphotyrosine and to study whether this treatment induced the cytoskeleton remodeling leading to the formation of podosomes. Indeed, orthovanadate (10–100 μM) induced in a time‐and dose‐dependent manner the redistribution of F‐actin and the formation of podosomes in BHK cells. Cytoskeleton remodeling occurred along with a marked increase of tyrosine phosphorylatcd proteins. The vanadate effect on the cytoskeletal phenotype was enhanced by the simultaneous treatment of cells with a phorbol ester. Under the latter conditions almost all BHK cells showed podosomes. The vanadate effect was reversible insofar as podosomes and tyrosine‐phosphorylated proteins disappeared. Then, vanadate treatment of normal cells induced the cascade of events leading to the cytoskeletal changes typical of transformation and suggested that the transformed cytoskeletal phenotype may he primarily induced by the tyrosine phosphorylation of unknown target(s) operated by endogenous kinases.
“Dynamic morphology system”: A method for quantitating changes in shape, pseudopod formation, and motion in normal and mutant amoebae of Dictyostelium discoideum
Authors: Soll David R. , Voss Edward , Varnum‐Finney Barbara , Wessels Deborah
Published: December 2004

An automated, video‐driven system was used to measure approximately 30 parameters of cell motion and accompanying changes in shape. This “Dynamic Morphology System” is based upon the Expertvision Motion Analysis System and is driven by a SUN computer. With the aid of this system, amoebic movement and shape changes were compared for vegetative wild‐type Dictyostelium discoideum amoebae and a motility mutant, Mo‐1. The measured parameters included speed, angle change, bearing, length, width, roundness, boundary flow, and curvature; and cell behavior was visualized monitoring amoebic tracks, difference pictures, and a newly developed ring expansion plot. Wild‐type cells remained elongated, moved continuously and retained polarity throughout migration. In contrast, Mo‐1 did not translocate, was round rather than elongated, formed bulges rather than elongated pseudopods, and exhibited no polarity. In contrast to the anterior f‐action distribution in wild‐type cells, f‐actin in Mo‐1 was distributed evenly as a shell just under the entire plasma membrane, a distribution consistent with the lack of polar cytoplasmic expansion.
Changes in glycoprotein fucosylation in a concanavalin A‐resistant variant of a human leukemia cell line (K562)
Authors: Bernier Louise G. , Sullivan Arthur K.
Published: December 2004

Previous studies from this and other laboratories have shown that variants of tumor cell lines can be selected for resistance to the lytic action of natural killer (NK) cells. One of these (K562‐Clone I), when made resistant to the toxic effects of Concanavalin A (Con A‐R1), regained its sensitivity to NK. Comparing the plasma membranes of Clone I and Con A‐R1, we observed (1) a very similar electrophoretic pattern of membrane glycoproteins identified by binding to the lectins Con A, WGA, PNA, and SBA; (2) an increase in binding of Ulex europaeus lectin to a group of glycoproteins from Con A‐R1 that were sensitive to treatment with fucosidase and N‐glycanase and that had a diffuse mobility ranging in apparent molecular weight from 30 to 200 kDa; and (3) a marked decrease in binding of an antibody reactive with the lactoneofucopentaose III antigen (Lewis x). This constellation of changes is an unusual pattern to follow Con A resistance and may point to a pathway of glyco‐sylation that a leukemic cell might use to modify its recognition by the NK mechanism.
Gene‐specific acquisition of hormonal responsiveness in rat liver during development
Authors: Johnson Alfred C. , Lee Kai‐Lin , Isham Kenneth R. , Kenney Francis T.
Published: December 2004

Cloned cDNAs were used in hybridization analyses to assess hormonal responsiveness of two similarly regulated genes in livers of late‐term fetal rats. Transcription of the tyrosine aminotransferase gene and of gene 33 (Lee et al.: J Biol Chem 260:16433–16438, 1985) is enhanced by glucocorticoids and by each of the usually antagonistic hormonal agents, insulin and cAMP, in adult liver, and that of both genes is developmentally activated at or just prior to birth. The mRNA of gene 33 was found to be significantly increased by each of the hormonal regulators in livers of fetuses treated in utero. Expression of the nearly silent aminotransferase gene in fetal liver was appreciably increased by cAMP but was refractory to control by either glucocorticoids or insulin; capacity of this gene to respond to insulin was not realized until several days postpartum. The data indicate specificity in the developmental acquisition of the capacity of individual genes to respond to hormonal regulators.
Specificity of the interaction between phosphatidylinositol 4,5‐bisphosphate and the profilin:actin complex
Authors: Lassing Ingrid , Lindberg Uno
Published: December 2004

Profilactin, the profiling:actin complex, which is present in large amounts in extracts of many types of eukaryotic cells, appears to serve as the precursor of microfilaments. It was reported recently that profilactin interacts specifically with phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2) (Lassing and Lindberg: Nature 314:472–474, 1985.) The present paper describes in detail the behaviour of profilactin and profilin in the presence of different types of phospholipids and neutral lipids under different conditions. PtdIns(4,5)P2 is the only phospholipid found so far which in the presence of 80 mM KC1 and at Ca2+ concentrations below 10−5 M effectively dissociates profilactin with the resulting polymerization of the actin. Phosphatidylinositol 4‐monophosphate exhibits some activity but phosphatidylinositol is inactive. Both calf spleen profilin and profilin from human platelets form stable complexes with PtdIns(4,5)P2 micelles. PtdIns(4,5)P2 is active also when incorporated together with other phospholipids in mixed vesicles.
Developmentally regulated compartmentalization of adenylate cyclase in Dictyostelium discoideum
Authors: Hagmann Jörg
Published: December 2004

Adenylate cyclase of aggregation phase Dictoystelium discoideum is activated by extracellular adenosine 3′, 5′‐cyclic monophosphate (cAMP), and the cAMP syn‐thesized is secreted. The distribution of the enzyme was determined in sucrose gradients loaded with whole cell lysates. Cell lysates prepared after 4.5 hr of starvation revealed membranes containing adenylate cyclase at 44% and 33% sucrose. The activity of the latter peak was detected in the presence of the detergent (CHAPS), 3‐(3‐cholamidopropyl) dimethylammonio‐3‐propanesulfonate, which inhibited the activity of the former to some extent. Adenylate cyclase activity of the 2 peaks differed with respect to solubility in CHAPS and their kinetics. The 44% sucrose region of the gradient contained the bulk of the plasma membranes, as judged by a cell surface glycoprotein marker (contact site A). The 33% peak is composed of small vesicular structures, as determined by electron microscopy. The distribution of adenylate cyclase activity detected in sucrose gradients shifted from the 33% to the 44% sucrose peak during development. In addition, the 44% peak became increasingly resistant to the inhibitory effect of CHAPS, Both changes were accelerated by extracellular cAMP, but only the latter was abolished when the production of endogeneous cAMP was inhibited by caffeine. Pulsing cells with cAMP overcame the inhibitory effect of caffeine.
Somatotropin antagonism of insulin‐stimulated glucose utilization in 3T3‐L1 adipocytes
Authors: Glenn Kevin C. , Rose Kathleen S. , Krivi Gwen G.
Published: December 2004

It is well established that somatotropin (GH) antagonizes insulin action in vivo and that supraphysiologic concentrations of GH frequently result in insulin resistance and glucose intolerance. However, the demonstration of an anti‐insulin activity by GH in vitro has been difficult. This study, therefore, set out to determine whether cultures of 3T3‐L1 adipocytes could be used to examine the anti‐insulin activity of GH. The ability of insulin to stimulate glucose utilization by 3T3‐L1 adipocytes increases approximately five‐fold during the first 4 days following treatment of the cells with a differentiation medium. It was found that glucose utilization in 3T3‐L1 adipocytes is regulated in a reciprocal fashion by insulin and GH. Bovine or human GH directly inhibit up to 50% of insulin‐stimulated [14C]‐glucose incorporation into lipids in a concentration‐dependent manner. The 3T3‐L1 sensitivity to GH appears to be at the maximum (50% inhibition of an insulin response) immediately following removal of the cells from the differentiation medium and remains essentially constant during the subsequent 4 days. The GH inhibition of insulin action does not appear to be due GH enhancement of cellular degradation of insulin, competitive binding of GH to the insulin receptor, or GH‐induced decrease in cell number. The 3T3‐L1 adipocyte system appears to be a sensitive and reliable in vitro model with which to study the molecular mechanisms involved in both GH antagonism of insulin action and development of hormone responsiveness during cellular differentiation into adipocytes.
Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: Partial, characterization and differentiation from the alkaline phosphatase activity
Authors: Pappas Peter W.
Published: December 2004

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hy‐drolyzes p‐nitrophenyl phosphate over a broad pH range. Acid phosphatasc activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2‐mercaptoethanol, and ethylenediaminetetra‐acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X‐100, while such treatment causes a significant loss of acid phosphalase activity. Both activities are nonspecific and hydrolyze a variety of phos‐phorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.
Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids
Authors: Miller R. W. , McRae D. G. , Al‐Jobore A. , Berndt W. B.
Published: December 2004

Bacteroids having a high level of respiration‐supported nitrogenase activity were isolated from nitrogen‐fixing alfalfa root nodules. Gentle maceration under anaerobic conditions in the presence of sodium succinate and a fatty acid scavenging agent were employed in this method.

A large proportion of isolated bacteroids retained a triple membrane structure as shown by transmission electron microscopy. Dicarboxylic acids of the TCA cycle (malate, fumarate, succinate), but not glutamate or aspartate, supported sufficient respiratory activity to supply the nitrogenase system with ATP and reducing equivalents and to protect the nitrogenase system from inactivation by 4% oxygen over a period of 20–30 min. Sugars did not support nitrogenase activity in intact bacteroids. The properties of the isolated bacteroids were ascribed to minimal damage to the cytoplasmic membrane and peribacteroidal membrane during isolation.

With succinate as substrate and oxygen as terminal electron acceptor, initial nitrogenase activity was determined at 4% oxygen in the gas phase of the assay system employed. At this oxygen concentration, the sustained rate of acetylene reduction by respiring bacteroids was linear up to 30 min. Bacteroid activity declined rapidly with time of exposure to oxygen above 4% in the gas phase. The optimum temperature range for this activity was 10–20°C. Nitrogenase activity was measurable at incubation tempertures below 10°C under 4% oxygen. Functionally intact bacteroids had little nitrogenase activity under anaerobic conditions in the presence of an external source of ATP and reductant. Treatment of the bacteroids with chlorpromazine eliminated respirtation‐supported activity and rendered the bacteroid cell membrane permeable to external ATP. Bacteroids treated with chlorpromazine had high acetylene reducing activity with external ATP and dithionite in the absence of oxygen.
Immunofluorescent and immunochemical evidence for the expression of cytovillin in the microvilli of a wide range of cultured human cells
Authors: Pakkanen Raimo
Published: December 2004

We have previously purified a Mr 75,000 protein, cytovillin, from cultured human choriocarcinoma cells (JEG‐3) and shown that this protein was specifically confined to the microvillus membrane of these cells. I have now studied the expression and the subcellular distribution of cytovillin in eighteen normal and transformed human cell lines and strains by using immunoblotting and indirect immunofluorescence microscopy. In all cell types, cytovillin was highly enriched in cell surface protrusions. When cell types were ranked according to their staining intensity, choriocarcinoma was highest, then amniotic epithelial cells, other choriocarcinoma cells and tumor cells, and finally fibroblastoid cells. The latter only gave faint diffuse fluorescence on the plasma membrane and, occasionally, on the microvilli. However, detergent extracts of all cell types could be shown to contain cytovillin by the use of immunoblotting techniques. Metabolic pulse‐chase labelling experiments with JEG‐3 cells demonstrated synthesis of cytovillin as a single‐chain polypeptide. No precursor forms or specific proteolytic cleavage products could be seen either by immunoblotting or immunoprecipitation. The protein was found to be very stable with a biologic half‐life of about 25 hours. The pI determined by isoelectric focusing was 6.1. These results were consistent with cytovillin being an integral component of the microvilli and other surface extensions of all human cell types examined.
An immunological assessment of lysosomal enzymes and other macromolecules sulfated during vegetative growth of Dictyostelium discoideum
Authors: Davis Simon J. , Wheldrake John F. , Freeze Hudson H.
Published: December 2004

Western blotting and immunoprecipitation data indicated that lysosomal enzymes represent a subset of the sulfated macromolecules present in vegetative Dictyostelium discoideum amoebae and account for less than 2.5% of the total sulfate incorporated during vegetative growth. These data suggest that the majority of the highly sulfated macromolecules of vegetative D. discoideum amoebae are not related to the lysosomal enzymes.
Interferon and growth factor modulation of nuclear factors binding to 5′ upstream elements of the 2–5A synthetase gene
Authors: Williams Bryan R. G. , Rutherford Michael N. , Hannigan Gregory E.
Published: December 2004

We assayed fragments of the 5′ flanking sequence of the human 2–5A synthetase gene for their ability to respond to interferon‐α (IFN) and platelet‐derived growth factor (PDGF). Transient transfection assays identified a 40‐base pair fragment, which, regardless of orientation, could confer IFN‐inducibility on the thymidine kinase promoter. This same fragment was active in monkey and mouse cells and in the latter was responsive to PDGF. The effect of PDGF could be inhibited by anti‐interferon antibodies. Gel retardation assays, using the 40‐base pair probe, detected the presence of IFN‐modulated DNA‐binding factors in nuclear extracts from monkey cells. In mouse cells both IFN and PDGF induced the binding of nuclear factors to a synthetic 2–5A synthetase response sequence. Thus, both IFN and growth factors directly or indirectly modulate the binding of nuclear factors to the same region of the 2–5A synthetase gene.
Binding of cytochalasin B to platelets
Authors: Horne McDonald K.
Published: December 2004

The binding of cytochalasin B (CB) to human platelets and to isolated platelet cytosol and membranes has been analyzed with [3H]CB. High‐ and low affinity classes of saturable binding sites were associated with intact platelets. Binding at very low concentrations of CB (i.e., high‐affinity binding) was partially prevented by 100 mM D‐galatose or D‐glucose and to a much lesser extent by L‐glucose. Binding to platelet cytosol also involved two classes of sites with affinities and capacities similar to those observed with the whole cells. None of this binding, however, was affected by 100 mM D‐galactose. Saturable binding to platelet membranes occurred at sites with a uniform binding affinity. Approximately 52% of this binding was prevented by 1 M D‐galactose and another 15% by cytochalasin E (CE). We hypothesize that binding in the cytosol is to monomeric (low‐affinity) and polymerized (high‐affinity) actin, whereas membrane binding (high‐affinity only) occurs primarily at sites involved with galactose transport.
Effects of platelet‐derived growth factor and fibroblast growth factor on free intracellular calcium and mitogenesis
Authors: Tucker Robert W. , Chang David T. , Meade‐Cobun Kimberly
Published: December 2004

Although increased free intracellular calcium (Cai) may be one of the main regulators of cell growth and differentiation, studies in cell populations have implied that not all growth factors produce Cai increases. In order to examine in more detail whether Cai increases were related to mitogenesis, we used digital image analysis of intracellular Fura‐2 fluorescence to measure Cai in individual BALB/c 3T3 cells stimulated with either platelet‐derived growth factor (PDGF) or fibroblast growth factor (FGF). We found that PDGF induced larger and more prolonged Cai increases than FGF did, but that both growth factors induced an initial rapid increase in Cai (< 2 min) followed by a later sustained increase (> 20 min). Only the prolonged Cai increase required extracellular calcium. Following PDGF treatment (1–8 units/ml), the percentage of cells with a large peak Cai increase (> twofold) correlated with the percentage of cells made competent (subsequent growth in 1% platelet‐poor‐plasma). In contrast, purified bovine basic FGF (200–800 pg/ml) and recombinant human acidic FGF (10–300 ng/ml) produced peak Cai increases that were not directly correlated with mitogenesis. In addition, concentrations of intracellular Quin 2 that inhibited Cai transients also inhibited PDGF stimulation but not FGF stimulation of mitogenesis. Thus, Cai increases are necessary for mitogenesis in BALB/c 3T3 cells stimulated by PDGF, but not that stimulated by FGF.
Overexpression of the c‐erbB‐2 protein in human breast tumor cell lines
Authors: Hynes Nancy E. , Gerber Heinz A. , Saurer Suzanne , Groner Bernd
Published: December 2004

The c‐erbB‐2 proto‐oncogene is amplified in a high percentage of primary human breast tumors, suggesting that the overexpression of this gene may be involved in the development of human breast cancer. We have investigated five human breast tumor cell lines and have detected amplified c‐erbB‐2 gene copies in two of them. This amplification leads to overexpression of the c‐erbB‐2 protein. In addition, two other cell lines have elevated protein levels without gene amplification, suggesting that other mechanisms can lead to overexpression of the c‐erbB‐2 protein. These results are similar to those that we obtained during a study of primary breast tumors (Berger et al.: Cancer Res 48:1238–1243, 1988). These breast tumor cell lines should be useful for an analysis of c‐erbB‐2 expression and of the mechanisms that in some cases lead to overexpression.
Transforming growth factor β: Possible roles in the regulation of normal and leukemic hematopoietic cell growth
Authors: Keller Jonathan R. , Sing Garwin K. , Ellingsworth Larry R. , Ruscetti Francis W.
Published: December 2004

We have recently demonstrated that transforming growth factor (TGF)‐β1 and TGF‐β2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin‐3 (IL‐3) and human bone marrow by IL‐3 or granulocyte/macrophage colony‐stimulating factor (GM‐CSF) was inhibited by TGF‐β1 and TGF‐β2, while the proliferation of murine bone marrow by GM‐CSF or murine and human marrow with G‐CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF‐β1. In particular, CFU‐GM, CFU‐GEMM, BFU‐E, and HPP‐CFC, the most immature colonies, were inhibited by TGF‐β1, whereas the more differentiated unipotent CFU‐G, CFU‐M, and CFU‐E were not affected. TGF‐β1 inhibited IL‐3‐induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF‐β1 results in the resumption of normal growth. TGF‐β1 inhibited the growth of factor‐dependent NFS‐60 cells in a dose‐dependent manner in response to IL‐3, GM‐CSF, G‐CSF, CSF‐1, IL‐4, or IL‐6. TGF‐β1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythorid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF‐β, were also resistant to TGF‐β1 inhibition. These leukemic cells have no detectable TGF‐β1 receptors on their cell surface. Last, TGF‐β1 directly inhibited the growth of isolated Thy‐1‐positive progenitor cells. Thus, TGF‐β may be an important modulator of normal and leukemic hematopoietic cell growth.
Characterization of the membrane‐associated GTPase activity: Effects of chemotactic factors and toxins
Authors: Pelz Claudia , Matsumoto Tadashi , Molski Thaddeus F. P. , Becker Elmer L. , Sha'afi Ramadan I.
Published: December 2004

Membranes prepared from rabbit neutrophils exhibit GTPase activity which can be stimulated by the chemotactic factor fMet‐Leu‐Phe. The maximum contribution of the ATPase activities to the basal and the fMet‐Leu‐Phe‐stimulated GTPase activities are less than 20% and 9%, respectively. The basal GTPase activity has a Vmax = 34.2 ± 1.3 (pmol/mg protein, min) and a Km = 0.39 ± 0.03 μM; and the fMet‐Leu‐Phe‐stimulated has a Vmax = 52.3 ± 2.5 (pmol/mg protein, min), and a Km = 0.29 ± 0.02 μM. The GTPase activity can be stimulated by fMet‐Leu‐Phe and leukotriene B4. Unlike these two chemotactic factors, concanavalin A does not stimulate this GTPase activity. In addition, the rise in intracellular concentration of free calcium produced by concanavalin A is not inhibited by pertussis toxin treatment. Both the basal and stimulated GTPase activities are affected by pertussis toxin, cholera toxin and N‐ethylmaleimide.
Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions
Authors: Khandekar Sanjay S. , Jacobson Gary R.
Published: December 2004

Column chromatography of the Escherichia coli mannitol permease (mannitolspecific enzyme II of the phosphotransferase system) in the presence of deoxycholate has revealed that the active permease can exist in at least two association states with apparent molecular weights consistent with a monomer and a dimer. The monomeric conformation is favored by the presence of mannitol and by the phosphoenolpyruvate (PEP)‐dependent phosphorylation of the protein. The dimer is stabilized by inorganic phosphate (Pi), which also stimulates phospho‐exchange between mannitol and mannitol 1‐phosphate (a partial reaction in the overall PEP‐dependent phosphorylation of mannitol). Kinetic analysis of the phospho‐exchange reaction revealed that Pi stimulates phospho‐exchange by increasing the Vmax of the reaction. A kinetic model for mannitol permease function is presented involving both conformations of the permease. The monomer (or a less‐stable conformation of the dimer) is hypothesized to be involved in the initial mannitol‐binding and PEP‐dependent phosphorylation steps, while the stably associated dimer is suggested to participate in later steps involving direct phosphotransfer between the permease, mannitol and mannitol 1‐phosphate.