bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of fischer rat embryo cells
Authors: Duigou Gregory J. , Babiss Lee E. , Liaw Wen‐Shing , Zimmer Stephen G. , Ginsberg Harold S. , Fisher Paul B.
Published: December 2004

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild‐type 5 adenovirus (Ad5) or the Ela plus Elb transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host‐range Ad5 mutant. H5hrl, which contains a single base‐pair deletion of nucleotide 1055 in Ela resulting in a 28‐kd protein (calculated) in place of the wild‐type 51‐kd acidic protein, display a cold‐sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold‐sensitive Ad5 Ela mutants (H5dl 101 and H5in l06), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the Ela gene (0–4.5 m.u.) from H5hrl or H5dll01 also produce tumors in these animals. To directly determine the role of the 13S Ela encoded 289AA protein and the 12S Ela encoded 243AA protein in initiating an oncogenic phenotype in adenovirus‐transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975. which synthesizes the 289AA Ela protein but not the 243AA protein, and the Ad5 mutant H5dl 520 and the Ad2 mutant H2dl 1500, which do not produce the 289AA Ela protein but synthesize the normal 243AA Ela protein. All three types of mutant adenovirus‐transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus‐transformed CPEF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild‐type 13S Ela protein or a wild‐type 12S Ela protein when either protein is present alone, but does not occur when both wild‐type Ela proteins are present.
Structure and evolution of the human genes encoding protein C and coagulation factor IX
Authors: Long George L.
Published: December 2004

Human protein C is a vitamin K‐dependent plasma protein that serves as a feedback down‐regulator of the coagulation cascade by specifically degrading the protein cofactors VIIIa and Va. The protein C precursor consists of the following domains: leader peptide, “gla” region, two epidermal growth factor segments, and the activation peptide/serine protease. Comparison of amino acid sequences reveals that protein C and factor IX are homologous. A comparison of the genes for protein C and factor IX shows that all seven of the introns within the protein coding regions are in identical positions and correspond to protein structure‐function domain boundries. However, the base compositions of the two genes (coding and noncoding regions) are remarkably different: ∼60% guanine + cytosine (G + C) for protein C versus ∼40% G + C for factor IX. One possible explanation for this phenomenon is that the factor IX gene (located on the X chromosome) has undergone extensive deoxycytosine methylation and subsequent spontaneous deamination mutagenesis, resulting in a net C to thymine (and G to adenine) transition. This would suggest that the protein C gene may represent a more primitive form of the gene duplication precursor.
Tyrosyl and phosphatidylinositol kinases of human erythrocyte membranes
Authors: Vossler Mark R. , Coco Anna , Strausser Bimmie T. , Zaricznyj Christine , Macara Ian G.
Published: December 2004

The tyrosyl kinase and phosphatidylinositol (PI) kinase activities of human red cells have been partially purified and characterized. Although the PI kinase required detergent for solubilization, the major tyrosyl kinase of the red cell could be extracted by high salt. A very small residual activity remained associated with the membranes, however, that was solubilized with the PI kinase and copurified through an ammonium sulfate precipitation and diethylaminoethyl (DEAE) ion‐exchange step gradient elution. However, the two activities were found to differ with respect to their apparent Kms for ATP and Mg2+; they showed different half‐lives for temperature inactivation, possessed different relative activities in the presence of Mn+ and Ca2+, and were separable by elution from a DEAE‐Trisacryl ion exchange column using a linear NaCl gradient. The kinetic parameters of the membrane‐associated tyrosyl kinase differed from those of the salt‐extracted enzyme. PI kinase was not activated by pretreatment with the tyrosyl kinase p68v‐ros or by addition of the phosphotyrosyl phosphatase inhibitor, vanadate, to intact membranes, and was not competitively inhibited by the tyrosyl kinase substrate poly(Glu4,Tyr). We conclude that the human red cell phosphatidylinositol and tyrosyl kinases are distinct and separable activities, and that at least two separable tyrosyl kinases are present in human erythrocytes.
Genes that cooperate with tumor promoters in transformation
Authors: Colburn Nancy H. , Smith Bonita M.
Published: December 2004

Tumor‐promoting phorbol esters, like growth factors, elicit pleiotropic responses involving biochemical pathways that lead to different biological responses. Genetic variant cell lines that are resistant to mitogenic, differentiation, or transformation responses to tumor promoters have been valuable tools for understanding the molecular bases of these responses. Studies using the mouse epidermal JB6 cell lines that are sensitive or resistant to tumor promoter‐induced transformation have yielded new understanding of genetic and signal transduction events involved in neoplastic transformation. The isolation and characterization of cloned mouse promotion sensitivity genes pro‐1 and pro‐2 is reviewed. A new activity of pro‐1 has been identified: when transfected into human cancer prone basal cell nevus syndrome fibroblasts but not normal fibroblasts mouse pro‐1 confers lifespan extension on these cells. Recently, we have found that a pro‐1 homolog from a library of nasopharyngeal carcinoma, but not the homolog from a normal human library, is activated for transferring promotion sensitivity. The many genetic variants for responses to tumor promoters have also proved valuable for signal transduction studies. JB6 P‐ cells fail to show the 12‐O‐tetradecanoyl‐phorbol‐13‐acctate (TPA)‐induced synthesis of two proteins of 15 and 16 kD seen in P+ cells. P−, P+, and TPA transformed cells show a progressive decrease in both basal and TPA‐inducible levels of a protein kinase C substrate of 80 kD. P− cells are relatively resistant both to anchorage‐independent transformation and to a protein band shift induced by The calcium analog lanthanum. It appears that one or more calcium‐binding proteins and one or more pro genes may be critical determinants of tumor promoter‐induced neoplastic transformation.
Mode of estrogen action on cell proliferation in CAMA‐1 cells: II. Sensitivity of G1 phase population
Authors: Leung Benjamin S. , Potter Anne H.
Published: December 2004

The mammary cancer cell line CAMA‐1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate (1) the sensitivity of a specific cell cycle phase or phases to 17β‐estradiol (E2), (2) the effect of E2 on cell cycle kinetics, and (3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2‐induced 3H‐thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2‐treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2‐induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that (1) the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; (2) the E2 stimulation of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, (3) E2 does not facilitate faster cell division; and (4) estrogen‐induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2‐modulated Gl events related to tumor growth.
Expression of coronavirus E1 and rotavirus VP10 membrane proteins from synthetic RNA
Authors: Armstrong John , McCrae Malcolm , Colman Alan
Published: December 2004

Some viruses acquire their envelopes by budding through internal membranes of their host cell. We have expressed the cloned cDNA for glycoproteins from two such viruses, the E1 protein of coronavirus, which buds in the Golgi region, and VP10 protein of rotavirus, which assembles in the endoplasmic reticulum. Messenger RNA was prepared from both cDNAs by using SP6 polymerase and either translated in vitro or injected into cultured CV1 cells or Xenopus oocytes. In CV1 cells, the El protein was localised to the Golgi region and VP10 protein to the endoplasmic reticulum. In Xenopus oocytes, the E1 protein acquired post‐translational modifications indistinguishable from the sialylated, O‐linked sugars found on viral protein, while the VP10 protein acquired endoglycosidase‐H‐sensitive N‐linked sugars, consistent with their localisation to the Golgi complex and endoplasmic reticulum, respectively. Thus the two proteins provide models with which to study targeting to each of these intracellular compartments. When the RNAs were expressed in matured, meiotic oocytes, the VP10 protein was modified as before, but the E1 protein was processed to a much lesser extent than in interphase oocytes, consistent with a cessation of vesicular transport during cell division.
Regulation of sperm flagellar motility by calcium and cAMP‐dependent phosphorylation
Authors: Brokaw Charles J.
Published: December 2004

There is substantial evidence that cAMP‐dependent phosphorylation is involved in the activation of motility of spermatozoa as they are released from storage in the male reproductive tract. This evidence includes observations that in vivo activation of motility can be inhibited by protein kinase inhibitors, can be reversed by protein phosphatase treatment of demembranated spermatozoa, and is associated with phosphorylation of sperm proteins, and observations that spermatozoa that have not been activated in vivo can be activated in vitro by cAMP‐dependent phosphorylation. Activation in vivo can often be triggered by conditions that increase intracellular pH, but the relevance of this to in vivo activation under natural conditions and the steps between pH increase and cAMP increase have not been fully established. The relationships between changes in the protein substrates for cAMP‐dependent phosphorylation and changes in axonemal function are still unknown.

Sperm chemotaxis to egg secretions is widespread; in the sea urchin Arbacia, the egg jelly peptide resact has been identified as a chemoattractant. Response to chemoatlractants involves changes in assymmetry of flagellar bending waves, and similar changes in asymmetry can be produced in vitro by increases in [Ca++]. Temporal changes in resact receptor occupancy might lead to transient changes in intracellular [Ca++] and the asymmetry of flagellar bending, but many links in this hypothetical sequence remain to be established.

Both of these signalling systems offer immediate opportunities for investigations of biochemical pathways leading to easily assayable biological responses. However, complications resulting from interactions between these two systems need to be considered.
Membrane association of collagenase stimulatory factor(s) from B‐16 melanoma cells
Authors: Biswas Chitra , Nugent Matthew A.
Published: December 2004

Past studies have shown that contact between tumor cells and fibroblasts results in stimulation of collagenase production by the fibroblasts. Membrane fractions prepared by differential centrifugation of sonicated B‐16 melanoma cells were shown here to contain a collagenase stimulatory factor(s) (CSF). Trypsin treatment of intact B‐16 cells prior to membrane fractionation led to loss of 90% of the total activity, indicating that CSF is localized on the outer surface of the cells. Stimulation of fibroblast collegenase production was also observed with dialyzed octylglucoside extracts of the B‐16 membranes. Additional of exogeneous lipid, ie, a mixture of phosphatidylcholine and phosphatidylserine, to the detergent extract of the membranes followed by dialysis and centrifugation at 100,000g resulted in 80% recovery of the factor activity in the pellet containing reconstituted lipid vesicles. Fractionation of tritium‐labeled, reconstituted lipid vesicles on a Sephacryl S‐300 column revealed that the collagenase stimulatory factor coeluted with the radioactive lipid vesicles. The fractionated lipid vesicles lost stimulatory activity completely after trypsin treatment or heating at 65°C, indicating that the factor is a protein.
Identification of androgen response elements in mouse mammary tumour virus and the rat prostate C3 gene
Authors: Parker Malcolm G. , Webb Paul , Needham Maurice , White Roger , Ham Jonathan
Published: December 2004

The effect of steroid hormones on mouse mammary tumour virus (MMTV) promoter activity has been analysed in the breast cancer cell lines ZR‐75‐1 and T‐47D 11. Both androgens and progestins were stimulatory in both cell lines, whereas glucocorticoids were stimulatory in only ZR‐75 cells. A glucocorticoid response was restored to T‐47D cells by the transient expression of a glucocorticoid receptor expression vector, suggesting that endogenous receptors were limiting. The effect of androgen was mediated by a region of MMTV previously shown to be essential for glucocorticoid and progestin responses between nucleotides −201 and −69 upstream of the cap site.

Similarly, the effect of androgens on the rat prostate C3(l) gene promoter was analysed in the rat prostate cell line T5. Although these cells had functional androgen receptors which could stimulate MMTV‐promoter activity, they were incapable of interacting with androgen response elements to stimulate transcription from the C3(l) promoter
Pro‐opiomelanocortin gene: A model for negative regulation of transcription by glucocorticoids
Authors: Drouin Jacques , Charron Jean , Gagner Jean‐Pierre , Jeannotte Lucie , Nemer Mona , Plante Richard K. , Wrange Örjan
Published: December 2004

The gene encoding pro‐opiomelanocortin (POMC) offers an interesting model system to study negative control of transcription in eucaryotes. Indeed, glucocorticoid hormones specifically inhibit transcription of the POMC gene in the anterior pituitary. The POMC gene is predominantly expressed in the anterior and intermediate lobes of the pituitary. However, only anterior pituitary POMC transcription is inhibited by glucocorticoids and stimulated by corticotropino‐releasing hormone (CRH). Rat POMC promoter sequences required for anterior pituitary‐specific expression were localized between positions −480 and −34 base pairs (bp) by DNA‐mediated gene transfer into the POMC‐expressing tumor cells, AtT‐20. These POMC promoter sequences also confer glucocorticoid inhibition of transcription. While two of the six in vitro binding sites for purified glucocorticoid receptor identified in the rat POMC gene are within these sequences, only one is required for glucocorticoid inhibition; this binding site is located at position −63 bp in the promoter and overlaps a putative CCA AT box sequence. The DNA sequence of the POMC −63 bp receptor binding site is homologous to receptor binding sites identified in the glucocorticoid responsive element (GRE) of glucocorticoid‐inducible genes. However, DNA sequence divergencies between these sites, in particular within the conserved hexanucleotide sequence 5′‐TGTYCT‐3′, may be involved in their opposite transcriptional activity. Alternatively, binding of the receptor in the promoter proximal region of the POMC gene may inhibit transcription by a hormone‐dependent represser mechanism.
Characterization of a hepatic proliferation inhibitor (HPI): Effect of HPI on the growth of normal liver cells—comparison with transforming growth factor beta
Authors: Huggett Anthony C. , Krutzsch Henry C. , Thorgeirsson Snorri S.
Published: December 2004

Improvements in the purification of a hepatic proliferation inhibitor (HPI) from adult rat liver have yielded a product that has an inhibitory activity 1,000‐fold greater than previously reported. The growth inhibitory activity, which could be eluted from SDS‐PAGE at 17–19 kilodaltons (kD), was compared to that of transforming growth factor beta (TGF‐β). The ID50 of the HPI preparation in Fischer rat liver epithelial cells was 50 pg/ml (2.5 pM) compared to a value of 260 pg/ml (10.4 pM) obtained for pure human TGF‐β. Both inhibitors also modulated the stimulation of DNA synthesis in primary hepatocytes by either epidermal growth factor or a growth stimulatory activity prepared from serum of hepatectomized rats. The ID50S of HPI and TGF‐β in these cells were 250 pg/ml and 40 pg/ml, respectively. In contrast to TGF‐β the growth inhibitory activity of HPI was unaltered in the presence of an antibody raised against TGF‐β. The possible mechanism of action of HPI is discussed.
Uncoupling of translocation across microsomal membranes from biosynthesis of influenza virus hemagglutinin
Authors: Chao Chuck C.‐K. , Bird Phil
Published: December 2004

This communication presents our recent studies on the biosynthesis of influenza virus hemagglutinin (HA) in a mammalian‐cell‐free system and its translocation across microsomal membranes. RNAs coding for wild‐type (full‐length) and mutant (truncated) forms of HA were generated by in vitro transcription by using bacteriophage T7 DNA‐dependent RNA polymerase. These RNAs were translated in a rabbit reticulocyte system that was supplemented with dog pancreas membranes, either before translation was initiated or after it had been artificially terminated with the antibiotic cycloheximide. All forms of HA could be cotranslationally translocated. However, only truncated molecules (83% of full length) could translocate after protein synthesis had been terminated. Posttranslational translocation was dependent on the presence of a functional N‐terminal signal sequence and occurred only in the presence of ribosomes. The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed based on the signal hypothesis.
Invasive activity and chemotactic response to growth factors by Kaposi's sarcoma cells
Authors: Albini Adrians , Mitchell Charles D. , Thompson Erik W. , Seeman Ruth , Martin George R. , Wittek Alec E. , Quinnan Gerald V.
Published: December 2004

Kaposi's sarcoma (KS) is a relatively low grade neoplasm, classically occurring in the skin of elderly men. A more virulent and invasive form of Kaposi's sarcoma has been described in patients with acquired immune deficiency syndrome (AIDS). The origin and identification of the tumor cells in these lesions is controversial. Here we have studied the behavior of cells derived from KS lesions in an in vitro assay which measures the ability of cells to invade through a reconstituted basement membrane. In agreement with previous work, KS cells obtained under selective culture conditions were invasive showing activity comparable to that of malignant tumor cells. Normal fibroblasts, smooth muscle cells, and endothelial cells did not demonstrate invasive behavior under the same experimental conditions. To characterize further the nature of the KS cells we tested the chemotactic response of cells from the most invasive line to a variety of growth factors and compared their response to those of fibroblasts, smooth muscle, and endothelial cells. These studies suggest that normal cells respond to a unique repertoire of chemotactic factors. The chemotactic response of the KS cells most closely resembled that of smooth muscle cells and was quite distinct from endothelial cells. These results indicate that the KS‐derived cultures contain invasive cells with a smooth muscle cell‐like phenotype.
Aspects of signal transduction in stimulus exocytosis‐coupling in Paramecium
Authors: Satir Birgit H. , Busch Gerald , Vuoso Alice , Murtaugh Timothy J.
Published: December 2004

This paper deals with the detailed mechanisms of signal transduction that lead to exocytosis during regulative secretion induced by specific secretagogues in a eukaryotic cell, Paramecium tetraurelia. There are at least three cellular compartments involved in the process: (I) the plasma membrane, which contains secretagogue receptors and other transmembrane proteins, (II) the cytoplasm, particularly in the region between the cell and secretory vesicle membranes, where molecules may influence interactions of the membranes, and (III) the secretory vesicle itself.

The ciliated protozoan Paramecium tetraurelia is very well suited for the study of signal transduction events associated with exocystosis because this eukaryotic cell contains thousands of docked secretory vesicles (trychocysts) below the cell membrane which can be induced to release synchronously when trioggered with secretagogue. This ensures a high signal‐to‐noise ratio for events associated with this process. Upon release the trichocyst membrane fuses with the cell membrane fuses with the cell membrane and the trichocyst content undergoes a Ca2+‐dependent irreversible expansion. Secretory mutants are available which are blocked at different points in the signal transduction pathway.

Aspects of the three components mentioned above that will be discussed here include (a) the properties of the vesicle content, its pH, and its membrane; (b) the role of phosphorylation/dephosphorylation of a cytosolic 63‐kilodalton (kDa)Mr protein in membrane fusion; and (c) how influx of extracellular Ca2+ required for exocytosis may take place via exocytic Ca2+ channels which may be associated with specific membrane microdomains (fusion rosettes).
Electrophoretic analysis of four high molecular weight sialoglycoproteins produced by metastatic human colon carcinoma cells
Authors: Irimura T. , Carlson D. A. , Ota D. M.
Published: December 2004

We have found that polyacrylamide gel electrophoresis in 3% gels in the presence of sodium dodecyl sulfate is suitable for the separation of cellular glycoproteins having molecular weights ranging from 200,000 to 1,000,000. The gels secured on a rigid support (Gelbond) allow blotting techniques with lectins and antibodies for the detection of glycoproteins. Using these methods we have separated lysates of HT‐29 human colon carcinoma cells and detected at Jeast four distinct high molecular weight Sialoglycoproteins having molecular weights of 900,000, 740.000, 560,000, and 450,000. The expression of the 9000,000 component, as revealed by wheat germ agglutinin binding, was much higher in a subline of HT‐29 cells established from liver metastases in a nude mouse than it was in the parental cells. The relative intensity of wheat germ agglutinin binding to these four sialoglycoprotein components differs depending upon their growth phase in vitro. These glycoproteins were also detectable by the binding of peanut agglutinin, provided the glycoproteins were previously treated in the gels with mild acid to remove the sialic acid from their carbohydrate chains, suggesting that mucin‐type carbohydrate chains are present on these glycoproteins. The same set of glycoproteins can be detected by metabolic labeling of the cells with [3H] glucosamine in tissue culture. Very similar glycoprotein profiles are revealed by metabolic labeling of fresh colon carcinoma tissues with [3H] glucosamine in vitro.
Characterization of mono‐, di‐, and tri‐O‐acetylated sialic acids on human cells
Authors: Hutchins Jeff T. , Reading Christopher L.
Published: December 2004

The presence of mono‐, di‐, and tri‐O‐acetylated sialic acids on human cells was demonstrated by using radiochromatographic and chemical techniques. Human melanoma cells and fresh colon tissue were biosynthetically labeled with 6‐ (3H) glucosamine. Radiolabeled sialic acids were hydrolytically removed from cellular glycoconjugates, purified by ion‐exchange chromatography, and separated by paper chromatography on the basis of the number of O‐substitutions on each sialic molecule, This analytical technique characterized radiolabeled sialic acids that migrated with the same Rf as synthetic mono‐, di‐, and tri‐O‐acetylated 14C‐labeled sialic acids. The mono‐O‐acetylated sialic acids were characterized by their sensitivity to sodium periodate oxidation and a crude mouse liver esterase preparation. The di‐ and tri‐O‐acetylated sialic acids were characterized by their resistance to sodium periodate oxidation and sensitivity to the action of crude mouse liver esterase. Chromatographically separated di‐ and tri‐O‐acetylated sialic acids from normal human colon tissue were characterized by their respective ion molecular weights by using fast‐atom bombardment‐mass spectrometry. Using these methods, we chemically characterized mono, di‐, and tri‐O‐acetylated sialic acids expressed on human cells. Aberrant expression of O‐acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di‐ and tri‐O‐acetylated sialic acids.
Major O‐glycosylated sialoglycoproteins of human hematopoietic cells: Differentiation antigens with poorly understood functions
Authors: Gahmberg Carl G. , Autero Matti , Hermonen Jorma
Published: December 2004

All human hematopoietic cells seem to contain a major, heavily O‐glycosylated sialoglycoprotein. Glycophorin A is specific for the erythroid lineage of cells, and leukocytes have a major sialoglycoprotein, also called leukosialin or sialophorin. Cell differentiation results in patterns of O‐glycosylation in these proteins, which reflect the stage of differentiation within a cell lineage as well as lineage specificity. The altered carbohydrate compositions may influence the interactions of the cells with external ligands. Healthy individuals lacking glycophorin A in their red cells are known, whereas a deficiency of the leukocyte sialoglycoprotein may result in immunological disease. Although little is known about the physiological functions of these proteins, they form interesting models for studies on regulation of glyco‐sylation, biosynthesis of O‐glycosylated glyoproteins, and function of cell surface receptors.
Endogenous lectins as mediators of tumor cell adhesion
Authors: Lotan Reuben , Raz Avraham
Published: December 2004

Endogenous carbohydrate‐binding proteins have been found in various normal tissues and cells. Although lectins with different sugar‐binding specificities have been described, the most prevalent ones are those that bind β‐galactosides. The ability of some normal and malignant cells to bind exogenous carbohydrate‐containing ligands suggested that lectinlike activity is associated with the cell surface and that carbohydrate‐binding proteins might mediate intercellular recognition and adhesion. We found that extracts of various cultured murine and human tumor cells exhibit a galactoside‐inhibitable hemagglutinating activity. This activity was associated with two proteins of molecular weights of 34,000 and 14,500 daltons, which were purified by affinity chromatography by using immobilized asialofetuin. That these lectins are present on the cell surface was indicated by the binding of monoclonal antilectin antibodies to the surface of various tumor cells and by the immunoprecipitation of 125I‐labeled lectins from solubilized cell‐surface iodinated cells by polyclonal anti‐lectin antibodies. That these cell surface lectins are functional was demonstrated by the ability of the galactose‐terminating asialofetuin to enhance cell aggregation and of asialofetuin glycopeptides to block this homotypic aggregation as well as to suppress cell attachment to substratum, and by the inhibition of both asialofetuin‐induced cell aggregation and cell attachment to substratum by the binding of monoclonal antilectin antibodies to the cell surface. These findings implicate cell surface lectins as mediators of cell‐cell and cell‐substratum adhesion. Some of these cellular interactions might be important determinants of tumor cell growth and metastasis.
(2′‐5′)An‐dependent endoribonuclease: Enzyme levels are regulated by IFNβ, IFNγ, and cell culture conditions
Authors: Floyd‐Smith Georgia
Published: December 2004

The levels of a (2′‐5′) An‐dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2′‐5′)An, (2′‐5′)A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100–2,000 IRU IFNβ or IFNγ resulted in a similar 2–4‐fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2–3‐fold as cells approached saturation density. Serum‐starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFNβ or IFNγ. Regulation of RNase L levels by cell growth conditions as well as by IFNβ or IFNγ treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.
Cation‐independent mannose 6‐phosphate receptor contains covalently bound fatty acid
Authors: Westcott Keith R. , Rome Leonard H.
Published: December 2004

The cation‐independent mannose 6‐phosphate receptor (215,000 daltons) was isolated from embryonic bovine tracheal cells and embryonic human skin fibroblasts labelled with [3H]palmitic acid, the tritium label was detected in the protein upon fluorographic analysis of SDS‐polyacrylamide gels of the purified receptor. The label was not sensitive to hydroxylamine, methanolic KOH, or β‐mercaptoethanol, but labelled fatty acid was recovered from the protein by acidic methanolysis. Labelled receptor protein could not be isolated from cells grown in the presence of [3H]myristic acid. The results suggest the presence of amide‐linked palmitic acid in the structure of the cation‐independent mannose 6‐phosphate receptor.
Cytochemical, histological, and phylogenetic distribution of a 38,000‐dalton protein associated with transverse tubules
Authors: Tidball James G. , Gadus Michelle V.
Published: December 2004

A major protein in detergent extracts of skeletal muscle appears at 38,000 daltons in electrophoretic separations. Previous investigations have provided indirect evidence that a 38‐kD skeletal muscle protein is membrane associated, and this inference has served as the basis for speculations on 38‐kD protein function. In the present study, affinity purified, polyclonal antisera against 38‐kD protein from skeletal muscle are produced for immunolocalization and biochemical assays. Immunoblots of two dimensional electrophoretic separations show that this protein is heterogenously charged at pI ∼6.4. This 38‐kD protein is not extracted from muscle in low ionic strength or high ionic strength buffers, in isotonic buffers from pH 4 to pH 8 or in buffers containing 5 mM EGTA. The 38‐kD protein is extracted, however, by isotonic, pH 7.0 buffer containing 1.0% Triton‐X. Light microscope observations using indirect immunofluorescence of anti‐38‐kD labeled tissue show the protein distributed in a reticular pattern within cross‐sectioned muscle but not at the cell surface. Longitudinal sections show the protein concentrated in periodic, transverse bands. Purified fractions of muscle plasma membrane analyzed by immunoblotting contain 38‐kD protein. Immunoblots using anti‐38 kD show that this protein is present in all vertebrate skeletal muscle examined, however, the protein is present only in cardiac muscle that contains transverse tubules. The antibody does not recognize aldolase, another 38‐kD striated muscle protein.
Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross‐linking
Authors: Sinnett‐Smith James , Zachary Ian , Rozengurt Enrique
Published: December 2004

We have previously identified by chemical cross‐linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000–85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells [1]. Because bombesin‐like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000–85,000 cross‐linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000–85,000 affinity‐labelled band was the earliest cross‐linked complex detected in Swiss 3T3 cells incubated with 125I‐labelled gastrin‐releasing peptide (125I‐GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000–85,000 cross‐linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I‐GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I‐GRP binding and the affinity labelling of the Mr 75,000–85,000 protein. We also show that the cross‐linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000–85,000 complex applied to wheat germ lectin‐sepharose columns was cluted by addition of 0.3 M N‐acetyl‐D‐glucosamine. Second, treatment with endo‐β‐N‐actylglucosaminidase F reduced the apparent molecular weight of the affinity‐labelled band from 75,000–85,000 to 43,000, indicating the presence of N‐linked oligosaccharide groups.
Interferon‐α‐(IFN) producing CHO cell lines are resistant to the antiproliferative activity of IFN: A correlation with gene expression
Authors: van Heuvel M. , Govaert‐Siemerink M. , Bosveld I. J. , Zwarthoff E. G. , Trapman J.
Published: December 2004

CHO cell lines that constitutively produce the murine interferon‐α (IFN‐α) subspecies α4 and α6 were constructed. The producer cell lines were protected against viral (vesicular stomatitis virus) infection by the IFN species secreted, but were resistant to the growth inhibitory activity of the IFN species. As compared with α4, the α6 protein displayed a high antiproliferative activity when added to normal CHO cells, which correlates completely with the high antiviral activity of a6 on these cells. Three messenger ribonucleic acid (mRNA) species, which are normally induced in CHO cells by IFN treatment (1–8, 2–5A synthetase, and ISG 15) were constitutively present in CHO producer cell lines. The level of another mRNA (ISG 54), however, was very low in the producer cells as compared with its expression in short‐term IFN‐treated cells. These data indicate that 1–8, 2–5A synthetase and ISG 15 are not involved in the antigrowth activity of IFN in this system, but rather suggest a function of ISG 54 in this respect.
Tissue plasminogen activator and acute pulmonary embolism
Authors: Goldhaber Samuel Z. , Kessler Craig M. , Heit John , Markis John E. , Sharma G. V. R. K. , Dawley Douglas L. , Meyerovitz Michael F. , Vaughan Douglas E. , Parker J. Anthony , Come Patricia C. , Kim Ducksoo , Selwyn Andrew P. , Loscalzo Joseph , Braunwald Eugene
Published: December 2004

We assessed the efficacy and safety of peripheral intravenous recombinant human tissue‐type plasminogen activator (rt‐PA) in 47 patients with angiographically documented pulmonary embolism (PE). We administered 50 mg/2 h and, if necessary, an additional 40 mg/4 h. By 6 hours, 94% of the patients had angiographic evidence of clot lysis that was slight in 5, moderate in 12, and marked in 27 patients. Among the 34 patients with pulmonary hypertension prior to treatment, average pulmonary artery pressure decreased from 43/17 (27) to 31/13 (19) mm Hg (P < 0.0001). The average lung scan perfusion defect decreased from 37% before therapy to 16% (P < 0.01) after therapy among the 19 patients who had pre‐ and post‐treatment lung scans. Of 7 patients with pre‐ and post‐treatment imaging and Doppler echocardiograms, hypokinetic right ventricular wall movement (mild in 1, moderate in 2, and severe in 4) normalized in 5 and improved to mild hypokinesis in 2. Right ventricular diameter decreased from 3.9 ± 1.0 to 2.0 ± 0.5 cm (P < 0.005). Fibrinogen decreased 33% from baseline at 2 h and 42% from baseline at 6 h. However, patients with the greatest degree of angiographic clot lysis at 2 h had a preponderance of fibrinogenolysis over fibrinolysis, demonstrated by a lower ratio of cross‐linked fibrin degradation products to fibrin(ogen) degradation products (0.14 ± 0.09 vs. 0.54 ± 0.82) (P < 0.04). Among selected patients, peripheral intravenous rt‐PA is associated with rapid lysis of PE, improved pulmonary perfusion, and improved right ventricular function.
Cytotoxicity mediated by tumor necrosis factor in variant subclones of the ME‐180 cervical carcinoma line: Modulation by specific inhibitors of DNA topoisomerase II
Authors: Coffman Frederick D. , Green Lora M. , Godwin Angela , Ware Carl F.
Published: December 2004

The mechanism of tumor necrosis factor (TNF)‐induced cytotoxicity has been investigated using two clonal variants of the ME‐180 human cervical carcinoma cell line. The clonal lines were characterized with respect to their expression of TNF receptors, kinetics of cell death, and their ability to communicate intercellularly through gap junctions. The ME‐180.4 and ME‐180.8 clones were identified by their relative sensitivity to TNF induced lysis in a 24‐h assay. The dose of TNF required to kill 50% of the target cells was 60 pM for the sensitive ME‐180.4 and 2.5 nM for the ME‐180.8. However, when assay times were extended, the dose response for both clones was the same, indicating that a difference in the kinetics of cell death and not absolute TNF sensitivity existed between the ME‐180.4 and ME‐180.8 clones. Both clones were gap junction deficient as judged by their inability to transfer Lucifer yellow or 6‐carboxyfluorescein, a characteristic phenotype of cells sensitive to cytotoxicity by TNF. The level of surface receptor expressed on these clones was nearly identical with a Kd = 0.3 nM and 5,000 binding sites per cell. Measurement of the kinetics of cell death revealed that the time between the addition of TNF and the onset of observed cell death (induction phase) was much shorter for the ME‐180.4 (32–55 h) than for the resistant ME‐180.8 (55–80 h). Mitomycin C, a DNA alkylating agent, significantly reduced the length of the induction phase for both clones, although the kinetic difference between the clones remained unchanged. Two epipodophyllotoxins, VP‐16 and VM‐26, which specifically inhibit the rejoining activity of DNA topoisomerase II, showed a 10–100‐fold synergistic effect when combined with TNF as shown by isobologram analysis. VM‐26 when added to the resistant ME‐180.8 clones decreased the length of induction phase and abolished the kinetic difference observed with the ME‐180.4 clone. These results indicate that the variance in the TNF response of these two clones was closely associated with DNA topoisomerase II, and suggest that this enzyme may play an important role in TNF mediated cytotoxicity.
Role for interleukin‐1 in the pathogenesis of hypersensitivity diseases
Authors: Dinarello Charles A. , Endres Stefan
Published: December 2004

Interleukin‐1 (IL‐1), a polypeptide product of various cells, is one of the key mediators of the body's response to microbial invasion, inflammation, immunological reactions, and tissue injury. IL‐1 is a prominent member of a group of polypeptide mediators now called “cytokines.” Current evidence suggests that IL‐1 is not produced in health but that any perturbation such as inflammation or even slight injury triggers the expression of IL‐1 genes. The biological effects of IL‐1 are manifested in nearly every tissue and organ. These include various proinflammatory effects such as increased production of arachidonate metabolites, synovial cell proteases, activation of basophils, eosinophils and neutrophils, endothelial cell adhesiveness, and stimulation of lymphocyte responses. Control of IL‐1 synthesis in certain diseases is often appropriate. Although corticosteroids reduce both the transcription and translation of IL‐1, we have recently investigated the effect of dietary supplementation with N‐3 (omega‐3) fatty acids in human volunteers. The results indicate that increasing the amount of N‐fatty acids in the diet decreases the ability of blood mononuclear cells to synthesize IL‐1 in vitro. It is suggested that the ameliorative effects of N‐3 fatty acid dietary supplements in patients with hypersensitive diseases may be, in part, the result of decreased IL‐1 production.
Modulation of IL‐4‐induced human IgE production in vitro by IFN‐γ and IL‐5: The role of soluble CD23 (s‐CD23)
Authors: Pène J. , Chrétien I. , Rousset F. , Brière F. , Bonnefoy J. Y. , de Vries J. E.
Published: December 2004

IL‐4 specifically induced IgE production by peripheral blood lymphocytes or by tonsil or spleen cells from healthy donors. IL‐4‐induced IgE synthesis was dependent on CD4+ T cells and monocytes and was blocked by IFN‐γ, IFN‐α, and prostaglandin E‐2 (PGE‐2). These substances also inhibited IL‐4‐induced CD23 expression and subsequent release of soluble CD23 (s‐CD23). In addition, IgE production was blocked by F(ab′)2 fragments of an mAb against CD23. In contrast, IL‐5 enhanced IL‐4‐induced IgE production, provided IL‐4 was added at nonsaturating concentrations. This increase in IgE production correlated quantitatively with an enhanced release of s‐CD23. Collectively, these results indicate that there is a correlation between s‐CD23 release and IgE production. However, s‐CD23 fractionated from supernatants of the lymphoblastoid cell line RPMI‐8866 was ineffective in inducing IgE production in the absence of IL‐4, but acted synergistically with suboptimal concentrations of IL‐4. In addition, it is demonstrated that alloreactive T‐cell clones produced varying concentrations of IL‐4, IL‐2, or IFN‐γ upon stimulation. Only supernatants of 2/4 of these T‐cell clones induced a low degree of IgE synthesis, but in the presence of anti‐IFN‐γ antibodies, all four supernatants induced a strong induction of IgE production. This IgE synthesis was blocked specifically by anti‐IL‐4 antibodies, indicating that IL‐4 is the sole inducer of IgE synthesis. Our findings demonstrate that IL‐4‐induced IgE production involves complex interactions of T cells, B cells, and monocytes and is positively modulated by IL‐5 and s‐CD23 but down‐regulated by IFN‐γ, IFN‐α, and PGE‐2, respectively.
An expression system for trypsin
Authors: Vasquez John R. , Evnin Luke B. , Higaki Jeffrey N. , Craik Charles S.
Published: December 2004

The eukaryotic serine protease, rat anionic trypsin, and various mutants created by site‐directed mutagenesis have been heterologously expressed in Escherichia coli. The bacterial alkaline phosphatase (phoA) promoter was used to control the expression of the enzymes in an induced or constitutive fashion. The DNA coding for the eukaryotic signal peptide of pretrypsinogen was replaced with DNA coding for the phoA signal peptide. The phoA signal peptide successfully directs the secretion of the mammalian trypsinogen to the periplasmic space of E. coli. Active trypsin was expressed in the periplasm of E. coli by deleting the DNA coding for the activation hexapeptide of the zymogen. The activity of trypsin in the periplasm suggests that the enzyme is correctly activated and has folded such that the 12 cysteine residues involved in the six disulfide bonds of rat anionic trypsin have paired correctly.

A transcription terminator increased the level of expression by a factor of two. However, increasing the copy number of the plasmid decreased the levels of expression. Localization of the active enzyme in the periplasm allows rapid screening of modified trypsin activities and facilitates the purification of protein to homogeneity and subsequently to crystallinity.
Neoplastic modulation of extracellular matrix: Stimulation of chondroitin sulfate proteoglycan and hyaluronic acid synthesis in co‐cultures of human colon carcinoma and smooth muscle cells
Authors: Iozzo Renato V. , Sampson Phyllis M. , Schmitt Gail K.
Published: December 2004

Previous studies have shown that human colon carcinomas contain elevated amounts of chondroitin sulfate proteoglycan (CS‐PG) and hyaluronic acid, and that the major site of synthesis of these products is the host mesenchyme surrounding the tumor. These findings have led to the proposal that the abnormal formation of the tumor stroma is modulated by the neoplastic cells. The experiments of this paper were designed to explore further this complex phenomenon in an in vitro system using co‐cultures of phenotypically stable human colon smooth muscle (SMC) and carcinoma cells (WiDr). The results showed a 3–5‐fold stimulation of CS‐PG and hyaluronic acid biosynthesis in the co‐cultures as compared to the values predicted from the individual cell type cultured separately. The increase in CS‐PG was not due to changes in specific activity of the precursor pool, but was rather due to a net increase in synthesis, inasmuch as it was associated with neither a stimulation of cell proliferation nor with an inhibition of intracellular breakdown. These biochemical changes were corroborated by ultrastructural studies which showed a marked deposition of proteoglycan granules in the co‐cultures. Several lines of evidence indicated that the SMC were responsible for the overproduction of CS‐PG: (i) SMC synthesized primarily CS‐PG when cultured alone, in contrast to the WiDr, which synthesized exclusively heparan sulfate proteoglycan; (ii) only the SMC in co‐culture stained with an antibody raised against the amino terminal peptide of a CS‐PG (PG‐40), structurally and immunologically related to that synthesized by the SMC; (iii) the stimulation of CS‐PG in SMC could be reproduced, though to a lesser extent, using medium conditioned by WiDr, whereas medium conditioned by SMC had no effects on WiDr. In conclusion this study has reproduced in vitro a tumor‐associated matrix with a proteoglycan composition similar to that observed in vivo and provides further support to the concept that production of a proteoglycan‐rich extracellular environment is regulated by specific tumor‐host cell interactions.