bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformation by the v‐fms oncogene product: An analog of the CSF‐1 receptor
Authors: Rettenmier Carl W. , Jackowski Suzanne , Rock Charles O. , Roussel Marline F. , Sherr Charles J.
Published: December 2004

The product of the c‐fms proto‐oncogene is related to, and possibly identical with, the receptor for the macrophage colony‐stimulating factor, M‐CSF (CSF‐1). Unlike the product of the v‐erbB oncogene, which is a truncated version of the EGF receptor, the glycoprotein encoded by the v‐fms oncogene retains an intact extracellular ligand‐binding domain so that cells transformed by v‐fms express CSF‐1 receptors at their surface. Although fibroblasts susceptible to transformation by v‐fms generally produce CSF‐1, v‐fms‐mediated transformation does not depend on an exogenous source of the growth factor, and neutralizing antibodies to CSF‐1 do not affect the transformed phenotype. An alteration of the v‐fms gene product at its extreme carboxyl‐terminus represents the major structural difference between it and the c‐fms‐coded glycoprotein and may affect the tyrosine kinase activity of the v‐fms‐coded receptor. Consistent with this interpretation, tyrosine phosphorylation of the v‐fms products in membranes was observed in the absence of CSF‐1 and was not enhanced by addition of the murine growth factor. Cells transformed by v‐fms have a constitutively elevated specific activity of a guanir.c nucleotide‐dependent, phosphatidylinositol‐4,5‐diphosphate‐specific phospholipase C. We speculate that the tyrosine kinase activity of the v‐fms/c‐fms gene products may be coupled to this phospholipase C, possibly through a G regulatory protein, thereby increasing phosphatidylinositol turnover and generating the intracellular second messengers diacylglycerol and inositol triphosphatc.
Polypeptide changes associated with loss of proliferative potential during the terminal event in differentiation
Authors: Wier Marjorie L. , Scott Robert E.
Published: December 2004

The differentiation of murine mesenchymal stem cells occurs in nonterminal and terminal phases. In previous reports we established the characteristics of nonterminally differentiated cells and showed that transition from the nonterminal to the terminal state of differentiation can be induced by human plasma. We also showed that this transition is blocked by protein synthesis inhibitors and other pharmacological agents. In this paper, we have employed two‐dimensional gel electrophoresis to evaluate changes in specific polypeptides that arc induced when cells lose proliferative capacity associated with the terminal event in differentiation. Using silver staining procedures for analysis of electrophoretograms, we detected only seven major polypeptide differences between nonterminally differentiated and terminally differentiated cells. Six polypeptides were expressed only in preparations of terminally differentiated cells; these included two polypeptides identified in cytosolic fractions and four polypeptides identified in nuclear fractions. One polypeptide was also found to be selectively expressed only in nuclear fractions of nonterminally differentiated cells. Based on these observations we conclude that the loss of proliferative potential that occurs during the terminal event in mesenchymal stem cell differentiation is associated with changes in the composition of a limited number of specific polypeptides. We suggest that one or more of these polypeptides may be important in the regulation of cellular proliferation.
Oncogene amplification and chromosomal abnormalities in small cell lung cancer
Authors: Ibson J. M. , Waters J. J. , Twentyman P. R. , Bleehen N. M. , Rabbitts P. H.
Published: December 2004

Twelve cell lines isolated from patients with small cell lung cancer have been studied for amplification of the three characterised members of the myc proto‐oncogene family (c‐myc, N‐myc, and L‐myc) and for abnormalities of chromosome 3. Ten of these lines were being studied for the first time. Ten of the 12 small cell lung cancer cell lines had amplification of one member of the myc proto‐oncogene family. Amplification of c‐myc was observed in only one small cell lung line—a “morphological variant.” One “classic” small cell lung cancer line expressed c‐myc but had no obvious amplification of the gene. N‐myc and L‐myc were more commonly amplified than c‐myc. Chromosomal abnormalities (mainly deletions) in chromosome 3 were observed in all small cell lung carcinoma cell lines examined. When the small cell lung carcinoma lines were grouped according to “classic” or “variant” characteristics, it was found that the “classics” had deletions of the short arm of chromosome 3, whereas the “biochemical variants” had deletions of the long arm of chromosome 3. The extent of the deletions varied between cell lines. For the deletion in the short arm of chromosome 3 the minimum common region of overlap was assigned to bands 3p23–3p24.
Identity of common phosphoprotein substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction
Authors: Evans Stuart W. , Farrar William L.
Published: December 2004

Interleukin 2 (IL 2) is a polypeptide growth factor essential for the proliferation and differentiation of T lymphocytes, large granulocytic lymphocytes, and, potentially, cells of the antibody‐producing lineage, B lymphocytes. Many of the biological properties of IL 2 may be mimicked or potentiated by a potent class of tumor promoters, phorbol esters. Phorbol esters have recently been shown to associate with and activate a unique phospholipid/Ca2+‐dependent phosphotransferase, protein kinase C (PK‐C). Utilizing two‐dimensional gel electrophoresis, we have compared the IL 2 and diacylglycerol‐induced protein phosphorylation patterns of several IL 2‐dependent murine cell lines. Both IL 2 and synthetic diacylglycerol, 1‐oleyl‐2‐acetyIglycerol (OAG), stimulated phosphorylation of a number of protein substrates in intact cells compared to unstimulated controls. Three groups of substrates were identified; the first showed increased phosphorylation following stimulation with either IL 2 or OAG, while the second and third groups showed increased phosphorylation following stimulation with IL 2 but not OAG, and with OAG but not IL 2, respectively. Here, we characterize the kinetics of phosphorylation of one cellular substrate, p68, which appears to be phosphorylated in response to direct activators of PK‐C or lymphoid or myeloid growth factors in their respective lineage cell lines. The observation that IL 2 also stimulates a unique series of phosphoproteins in addition to those induced by direct PK‐C activators suggests that IL 2 may initiate additional protein kinase activities, unrelated to PK‐C, which may also be critical for the ligand‐receptor signal transduction process regulating growth and gene expression.
Soluble 80‐kd fragment of cell‐CAM 120/80 disrupts cell‐cell adhesion
Authors: Wheelock Margaret J. , Buck Clayton A. , Bechtol Kathleen B. , Damsky Caroline H.
Published: December 2004

Calcium‐dependent cell adhesion molecules (CAMs) mediate intercellular adhesion in epithelial cells and in preimplantation mammalian embryos. One of these molecules, cell‐CAM 120/80, is found on cells as a 120‐kd membrane glycoprotein and as a soluble 80‐kd species in conditioned culture medium [Damsky et al: Cell 34:455, 1983]. We have purified to homogeneity the soluble 80‐kd fragment of cell‐CAM 120/80 by using monoclonal antibody affinity chromatography. We have shown that the purified molecule can disrupt cell‐cell adhesion in cultured epithelial cells, thus indicating that it is directly involved in the adhesive process. In addition, we have further characterized both the 120‐kd cell‐associated molecule and its 80‐kd fragment, including N‐terminal sequence analysis.
Immunohistochemical demonstration of proteinase inhibitor alpha‐1‐antichymotrypsin in normal human central nervous system
Authors: Justice Deanna L. , Rhodes Roy H. , Tökés Zoltán A.
Published: December 2004

The presence of alpha‐1‐antichymotrypsin, a serine proteinase inhibitor with a high affinity for cathepsin G, is demonstrated in the normal human central nervous system (CNS) by immunohistochemical techniques. Paraffin‐embedded normal human CNS tissue from five adult, two fetal, one neonatal and three newborn autopsies were stained with monospecific rabbit antibodies to human alpha‐1‐antichymotrypsin using biotinylated goat anti‐rabbit antibodies and an avidin‐biotin‐ peroxidase complex. Positive immunostaining was seen in neurons and glial cells in the cerebral cortex, basal ganglia, hippocampus, cerebellum, brainstem, and spinal cord of the adults. The epithelium of the adult choroid plexus had the most intense staining in apical granular organelles corresponding in position to lysosomes or secretory granules. Ependymal cells, particularly those near the choroid plexus, were immunostained. The fetal CNS had no alpha‐1‐antichymotrypsin staining. Limited staining of choroid plexus, ependyma. and frontal lobe was found in the newborns. Immunostaining in the neonatal temporal lobe was only found in the choroid‐plexus epithelium. These observations establish a widespread distribution of this proteinase inhibitor in the normal human CNS. Developmental regulation of this inhibitor in the human CNS is also indicated.
Phosphatidylinositol cycle and its possible involvement in the regulation of cytoskeleton‐membrane interactions
Authors: Burn Paul
Published: December 2004

Receptor‐mediated activation of many cells, including blood platelets, leads to changes at the cytoplasmic side of the membrane. In platelets, phospholipases, such as phospholipase C and phospholipase A2, have been shown to become activated. From phospholipids they generate the second messengers diacylglycerol and inositol phosphate(s) and fatty acids, respectively. At the same time, actin polymerization and reorganization of actin filaments into bundles and networks occurs. Here, the association of lipids, radiolabeled either with saturated (palmitic acid) or unsaturated (arachidonic acid) fatty acids, with the cytoskeletons of resting and activated human blood platelets was studied. The relative binding of lipid components to the cytoskeleton of activated platelets labeled with palmitic acid is six times higher than that of platelets labeled with arachidonic acid. Analysis of lipids associated with isolated cytoskeletons of resting and activated platelets (labeled with palmitic acid) showed a 30‐fold increase in the binding of labeled lipids to the cytoskeletal structures during activation. Both diacylglycerol and fatty acids were found to be associated with the cytoskeleton of activated platelets. Gel filtration, chromatofocusing, and immunoprecipitation studies demonstrated tight binding of these lipids to α‐actinin. α‐Actinin is one of the proteins that rapidly becomes associated with the cytoskeleton during platelet aggregation; it is also one of the molecules proposed to act as an actin‐membrane linker. The results, reported indicate a possible participation of α‐actinin, fatty acids, and the phosphoinositide‐derived second messenger diacylglycerol in the regulation of cytoskeleton‐membrane interactions. Together with the results of others they suggest a possible involvement of the phosphatidylinositol cycle in the assembly of actin filaments and their anchoring to membranes.
A domain of synapsin I involved with actin bundling shares immunologic cross‐reactivity with villin
Authors: Petrucci Tamara C. , Mooseker Mark S. , Morrow Jon S.
Published: December 2004

Synapsin I is a neuronal phosphoprotein that can bundle actin filaments in vitro. This activity is under phosphorylation control, and may be related to its putative in vivo role of regulating the clustering and release of small synaptic vesicles. We have compared human and bovine synapsin I by peptide mapping, and have used NTCB (2‐nitro‐5‐thiocyano benzoic acid) cleavage to generate a series of peptide fragments from bovine synapsin I. After chymotryptic digestion, 88% of the tyrosine‐containing fragments appear to be structurally identical in human and bovine synapsin I, as judged by their positions on high‐resolution two‐dimensional peptide maps. The alignment of the NTCB peptides within the parent protein have been determined by peptide mapping, and the ability of these fragments to precipitate with actin bundles has been measured. Only peptides that are derived from regions near the ends of the protein are active. One such 25‐kDa peptide which sediments with actin also cross‐reacts with antibodies to chicken villin, an actin binding and bundling protein derived from the intestinal microvillus. Since in other respects villin appears to be an unrelated protein, these results suggest the possibility that certain actin binding proteins may show immunologic cross‐reactivity due to convergent evolution within the acting binding domain.
Structure‐function studies on Acanthamoeba myosins IA, IB, and II
Authors: Korn Edward D. , Atkinson Mark A. L. , Brzeska Hanna , Hammer John A. , Jung Goeh , Lynch Thomas J.
Published: December 2004

Myosins IA and IB are globular proteins with only a single, short (for myosins) heavy chain (140,000 and 125,000 daltons for IA and IB, respectively) and are unable to form bipolar filaments. The amino acid sequence of IB heavy chain shows 55% similarity to muscle myosins in the N‐terminal 670 residues, which contain the active sites, and a unique 500‐residue C‐terminus highly enriched in proline, glycine, and alanine. The C‐terminal region contains a second actin‐binding site which allows myosins IA and IB to cross‐link actin filaments and support contractile activity. Myosins IA and IB are regulated solely by phosphorylation of one serine on the heavy chain positioned between the catalytic site and the actin‐binding site that activates ATPase.

Myosins II is a more conventional myosin in composition (two heavy chains and two pairs of light chains), heavy chain sequence (globular head 45% identical to muscle myosins and a coiled‐coil helical tail), and structure (bipolar filaments). The tail of myosin II is much shorter than that of other conventional myosins, and it contains a 25 amino acid sequence in which helical structure is predicted to be weak or absent. The position of this sequence corresponds to the position of a bend in the monomer. Myosin II heavy chains also have a 29‐residue nonhelical tailpiece which contains three regulatory, phosphorylatable serines. Phosphorylation at the tip of the tail regulates ATPase activity in the globular head apparently through an effect on filament structure.
Simultaneous transfer of tumorigenic and metastatic phenotypes by transfection with genomic DNA from a human cutaneous squamous cell carcinoma
Authors: Ananthaswamy Honnavara N. , Price Janet E. , Goldberg Leonard H. , Bales Elise S.
Published: December 2004

High‐molecular‐weight genomic DNA isolated from a human cutaneous squamous cell carcinoma (AS) was assayed for its ability to induce tumorigenic transformation of NIH 3T3 cells. Subcutaneous injection of NIH 3T3 cells cotransfected with DNAs from AS tumor and pSV2‐neo plasmid not only induced tumors at the site of injection, but also metastasized spontaneously to the lungs in 100% of nude mice injected. DNA isolated from a representative primary tumor and a metastasis was again used in a second round of transfection. Injection of secondary transfectants into nude mice again resulted in induction of both subcutaneous tumors and spontaneous long metastases. Southern blot hybridization with ras‐specific probes revealed that DNA from both primary tumors and metastases induced by AS tumor DNA contained highly amplified Ha‐ras oncogene. Furthermore, DNAs from secondary tumors and metastases induced by DNA from a primary tumor and a metastasis also contained similar highly amplified Ha‐ras oncogene. These results suggest that the amplified Ha‐ras oncogene may be responsible for induction of both tumorigenic and metastatic phenotypes in NIH 3T3 cells transfected with DNA from AS tumor.
Posttranslational modification of ras proteins: Detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification
Authors: Tamanoi F. , Hsueh E. C. , Goodman L. E. , Cobitz A. R. , Detrick R. J. , Brown W. R. , Fujiyama A.
Published: December 2004

Products of ras genes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttranslational modification by fatty acid. In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification of ras proteins prior to the fatty acid acylation. The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel. The fatty acid acylation does not contribute to this mobility shift. This modification is affected by the dpr1 mutation which has recently been shown to affect the processing of yeast RAS proteins. To further characterize the nature of the modification event, we have cloned DPR1 gene from the DNA of Saccharomyces cerevisiae. The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb. Genes related to the DRP1 appear to be present in a distantly related yeast, Schizosaccharomyces pombe as well as in guinea pig and human cells.
Gene expression and tumor cell escape from host effector mechanisms in murine large cell lymphoma
Authors: LaBiche Ronald A. , Yoshida Mitsuzi , Gallick Gary E. , Irimura Tatsuro , Robberson Donald L. , Klostergaard Jim , Nicolson Garth L.
Published: December 2004

Using in vivo selection methods, we obtained metastatic sublines of the murine RAW 117 large cell lymphoma that form multiple liver metastases. The highly metastatic subline RAW117‐H10 has a low number of gp70 molecules expressed at the cell surface and low cytostatic sensitivity to activated syngeneic macrophages. This subline was infected with endogenous RNA tumor virus isolated from a high virus‐expressing RAW117‐P subline of low metastatic potential. After superinfection the H10 subline gradually increased its expression of cell surface gp70 and showed enhanced sensitivity to macrophage‐mediated cytostasis, suggesting that gp70 might be involved in host macrophage‐mediated surveillance. Culture of RAW117‐P and H10 cells in media conditioned by activated macrophages indicated that parental cells are severely growth inhibited in a dose dependent fashion while H10 cells showed almost no effect. Examination of differentially expressed genes in the highly metastatic RAW117‐H10 cells by analysis of RNA blots indicated that a mitochondrial gene was expressed at a level that was ∼ 10 times higher in H10 cells than in parental cells. This gene was identified as ND5, which codes for a subunit of NADH dehydrogenase (complex I of the mitochondrial electron transport chain); this complex is the target for an activated macrophage‐released cytostatic factor. Among other possibilities, the results are consistent with the suggestion that highly metastatic RAW 117 cells may escape macrophage surveillance by decreasing the synthesis of specific cell‐surface receptors for cytostatic molecules and increasing the synthesis of specific cellular targets for such molecules.
Comparison of N‐acetylglucosaminyltransferase V activities in Rous sarcoma‐transformed baby hamster kidney (RS‐BHK) and BHK cells
Authors: Arango Juan , Pierce Michael
Published: December 2004

Recent studies have desmonstrated that Rous sarcoma virus‐transformed baby hamster kidney (RS‐BHK) cells express twofold higher levels of those N‐linked oligosac‐charides that contain the sequence [GlcNAc‐β(1,6)Man (1,6)] compared to nontrans‐formed parental BHK cells (Pierce and Arango, J. Biol. Chem. 261, 10772 [1986]). We have investigated in RS‐BHK and BHK cells the activity of UDP‐GlcNAc:α‐D‐mannoside β(l,6)N‐acetylglucosaminyltransferase V, the enzyme that begins the synthesis of the sequence that is increased in the RS‐BHK cells. We have measured GnT V activity using UDP‐ [3H]‐ GlcNAc and a synthetic oligosaccharide acceptor, GlcNAcβ(1,2)Man α(1,6)Manβ‐O‐ (Ch2)8COOCH3, separating the radioactive product by a newly devised reverse‐phase chromatographic technique. Assayed under optimal conditions, the specific activity of GnT V is about fourfold higher in RS‐BHK sonicates than in BHK sonicates, suggesting that this increase in activity may be the primary mechanism that causes the increase in [GlcNAcβ(1,6) Man] sequences in the RS‐BHK cells. The apparent Km, values of the enzymes in RS‐BHK and BHK cell sonicates for UDP‐GIcNAc and the synthetic acceptor are similar, as are the pH optima. These results suggest that the increase in GnT V‐specific activity in RS‐BHK cells is not caused by the presence in these cells of a GnT V with markedly different kinetic properties.
Extracellular matrix receptors, ECMRII and ECMRI, for collagen and fibronectin correspond to VLA‐2 and VLA‐3 in the VLA family of heterodimers
Authors: Takada Yoshikazu , Wayner Elizabeth A. , Carter William G. , Hemler Martin E.
Published: December 2004

The Very Late Activation Antigen (VLA) proteins are a family of five related heterodimers, which also are part of the integrin superfamily of cell adhesion molecules. Except for the identification of VLA‐5 as a fibronectin receptor structure, the functions of the VLA proteins have remained unclarified. In this paper, immuno‐precipitation experiments with both anti‐α and anti‐β subunit antibodies showed that the previously identified cell adhesion receptor for collagen, extracellular matrix receptor II (ECMRII), is equivalent to VLA‐2. At the same time a previously described multispecific cell adhesion receptor for collagen, fibroncclin, and laminin (ECMRI) has been shown to be identical to VLA‐3. Although the mAb 12F1 and P1H5 both recognized VLA‐2 (ECMRII), they appeared to define distinct epitopes on the α2 subunit. On the other hand, the mAb PIB5 and J143 recognized the α3 subunit of VLA‐3 (ECMRI) at or near the same site. Consistent with the collagen receptor functions of VLA‐2 (ECMRII) and VLA‐3 (ECMRI), anti‐VLA β antiserum blocked cell attachment to collagen.
Recombinant human granulocyte‐colony stimulating factor and recombinant human macrophage colony stimulating factor synergize in vivo to enhance proliferation of granulocyte‐macrophage, erythroid, and multipotential progenitor cells in mice
Authors: Broxmeyer Hal E. , Williams Douglas E. , Cooper Scott , Hangoc Giao , Ralph Peter
Published: December 2004

Combinations of low dosages of purified recombinant human (rh) macrophage colony stimulating factor (M‐CSF; also termed CSF‐1) and rh granulocyte‐colony stimulating factor (G‐CSF) were compared alone and in combination for their influence on the cycling rates and numbers of bone marrow and splenic granulocyte macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with iron‐saturated human lactoferrin (LF). LF was used to enhance detection of the stimulating effects of exogenously added CSFs. Concentrations of each CSF that were not active in vivo when given alone were active when given together, with the other CSF. The concentrations of rhM‐CSF and rhG‐CSF needed to increase progenitor cell cycling in the marrow and spleen were reduced by factors of 40‐200 when these CSFs were administered in combination with low dosages of the other CSF. At the concentrations of rhM‐CSF and rhG‐CSF tested, synergism was not noted on absolute numbers of progenitor cells or total nucleated cell counts per organ o circulating in the blood. These findings may have potential relevance when considered in a clinical setting where the CSFs might be used in combination with other biotherapy and/or chemotherapy.
Colony‐stimulating factor‐1 receptor (c‐fms)
Authors: Sherr Charles J. , Roussel Martine F. , Rettenmier Carl W.
Published: December 2004

The macrophage colony‐stimulating factor, CSF‐1 (M‐CSF), is a homodimeric glycoprotein required for the lineage‐specific growth of cells of the mononuclear phagocyte series. Apart from its role in stimulating the proliferation of bone marrow‐derived precursors of monocytes and macrophages, CSF‐1 acts as a survival factor and primes mature macrophages to carry out differentiated functions. Each of the actions of CSF‐1 are mediated through its binding to a single class of high‐affinity receptors expressed on monocytes, macrophages, and their committed progenitors. The CSF‐1 receptor (CSF‐1R) is encoded by the c‐fms proto‐oncogene, and is one of a family of growth factor receptors that exhibits an intrinsic tyrosine‐specific protein kinase activity. Transduction of c‐fms sequences as a viral oncogene (v‐fms) in the McDonough (SM) and HZ‐5 strains of feline sarcoma virus has resulted in alterations in receptor coding sequences that affect its activity as a tyrosine kinase and provide persistent signals for cell growth in the absence of its ligand. The genetic alterations in the c‐fms gene that unmask its latent transforming potential abrogate its lineage‐specific activity and enable v‐fms to transform a variety of cells that do not normally express CSF‐1 receptors.
Biological functions of serine proteases in mast cells in allergic inflammation
Authors: Katunuma Nobuhiko , Kido Hiroshi
Published: December 2004

Serine proteases in mast cell granules, such as chymase, atypical chymase, and tryptase, which are major proteins in the granules, may play important roles in the process of immunoglobulin E (IgE)‐mediated degranulation and in pathobiological alterations in tissues. Indeed, inhibitors of chymase, substrate analogs, and antichymase F(ab′)2, but not inhibitors of tryptase, markedly inhibited histamine release induced by IgE‐receptor bridging but not that induced by Ca ionophore. In contrast, inhibitors of metalloprotease inhibited histamine release induced not only by IgE‐receptor bridging but also by Ca ionophore. These results suggest that chymase and metalloprotease are involved at different steps in the process of degranulation. The extents of inhibition of histamine release were closely correlated with the amounts of the inhibitors of chymase accumulated in the granules. After degranulation, the released proteases may in part contribute to pathobiological alterations in allergic disorders through generations of C3a anaphylatoxin and thrombin by human and rat tryptase, respectively, and those of angiotensin n and a chemotactic factor of neutrophils by human and rat chymase, respectively. Moreover, chymase and atypical chymase from rat were shown to destroy type IV collagen, and human tryptase was found to hydrolyze various plasma proteins, such as fibrinogen and high‐molecular‐weight kininogen. The biological activities of tryptase and chymase from rat may be regulated by their dissociation from and association with trypstatin, an endogenous inhibitor of these proteases.
Pharmacological profile of the substituted beta‐lactam L‐659,286: A member of a new class of human PMN elastase inhibitors
Authors: Bonney R. J. , Ashe B. , Maycock A. , Dellea P. , Hand K. , Osinga D. , Fletcher D. , Mumford R. , Davies P. , Frankenfield D. , Nolan T. , Schaeffer L. , Hagmann W. , Finke P. , Shah S. , Dorn C. , Doherty J.
Published: December 2004

Human polymorphonuclear leukocyte elastase (PMN elastase) is inhibited by L‐659, 286 (7α‐methoxy‐8‐oxo‐3‐[[(1,2,5,6‐tetrahydro‐2‐methyl‐5,6‐dioxo‐1,2,4‐triaz‐in‐3‐yl)thio]methyl]‐5‐thia‐1‐aza‐6R‐bicyclo [4.2.O]oct‐2‐ene‐2‐pyrrolidine carboxamide‐5,‐dioxide) with a Ki of 0.4 μM. This inhibition is time‐dependent, rapid, and only slowly reversible, with a t1/2 of > 3 days at 25°C. L‐659, 286 is also highly selective for PMN elastase, as it does not inhibit thrombin, trypsin, papain, plasmin, chymotrypsin, or cathepsin G. L‐659, 286 administered intratracheally inhibits lung damage caused by administration via the same route of human PMN elastase into hamsters. In marmosets, L‐659, 286 is cleared from blood very rapidly after an intravenous injection but is recovered in bronchoalveolar lavage fluid for several hours after intratracheal administration.
Proteolytic regulation of neurite outgrowth from neuroblastoma cells by thrombin and protease nexin‐1
Authors: Cunningham Dennis D. , Gurwitz David
Published: December 2004

This review summarizes studies on the reciprocal regulation of neuroblastoma neurite outgrowth by thrombin and protease nexin‐1 (PN‐1). PN‐1 recently was shown to possess the same deduced amino acid sequence as the glial‐derived neurite‐promoting factor. The neurite outgrowth activity of PN‐1 depends on its ability to inhibit thrombin. Thrombin not only blocks the neurite outgrowth activity of PN‐1, but it also brings about neurite retraction in the presence of PN‐1. Thrombin also produces neurite retraction in the absence of PN‐1 and other regulatory factors. This suggests that its activity is due to a direct action on cells. The neurite retraction by thrombin depends on its proteolytic activity. It does not occur with the other serine proteases that have been tested, indicating that it is a specific effect and is not due to a general proteolytic effect that could detach neurites from the culture dish. Serum brings about neurite retraction in certain neuroblastoma cells and primary neuronal cultures; most of this activity is due to residual thrombin in the serum. Together, these results suggest that PN‐1 and thrombin (or a thrombin‐like protease) play a role in regulation of neurite outgrowth.
Augmented nuclease activity during cellular senescence in vitro
Authors: Dayton Mark A. , Nahreini Piruz , Srivastava Arun
Published: December 2004

The molecular correlates of the limited proliferative potential of normal human diploid fibroblasts and extensive single‐strand braks in the genomic DNA of these cells were examined by transfection analyses in which DNA replication could be uncoupled from DNA damage and repair. Both supercoiled (fmI), and restriction endonuclease‐cleaved, linear (fmIII) molecules of a well‐defined bacterial plasmid DNA, pBR322, were transfected into, and subsequently recovered from, early and late passage fibroblasts. Southern blot analysis revealed that fmI DNA was converted by random nicks into fmII DNA slightly more rapidly in late passage cells compared with cells at early passage. Similarly, fmII and fmIII DNAs also sustained multiple random nicks and no appreciable net religation of free ends of fmIII DNA could be detected at either passage. In addition, the efficiency of in vitro ligation of fmIII DNA recovered from late passage cells was also reduced, compared with that from early passage cells, as determined by Southern blotting. These data suggest that in the absence of DNA replication, a putative nuclease activity may contribute to DNA damage observed in senescent cells, which, in turn, may be causally related to their limited replicative potential.
Identification of tyrosine‐phosphorylated colony‐stimulating factor 1 (CSF‐1) receptor and a 56‐kilodalton protein phosphorylated in intact human cells in response to CSF‐1
Authors: Huhn Richard D. , Cicione Mary Elizabeth , Frackelton A. Raymond
Published: December 2004

Colony‐stimulating factor 1 (CSF‐1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF‐1, (the c‐fms protein) is a protein‐tyrosine kinase activated by the binding of CFS‐1, the role of phosphorylation of cellular proteins in CSF‐1 signal transduction is poorly understood. Therefore, we examined the CSF‐1‐stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c‐fms protein). BeWo cells were metabolically labeled with 32Pi, stimulated with recombinant human CSF‐1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170‐kd protein, followed closely by the phosphorylation of a 56‐kd protein, was observed in response to CSF‐1. The 170‐kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti‐fms serum, consistent with its identity as the CSF‐1 receptor. Although purified human macrophages that proliferate in culture in response to CSF‐1 are not generally accessible, CSF‐1 did stimulate the phosphorylation of a 56‐kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF‐1‐stimulated cellular protein phosphorylation.
The role of Asp‐49 and other conserved amino acids in phospholipases A2 and their importance for enzymatic activity
Authors: van den Bergh Carel J. , Slotboom Arend J. , Verheij Hubertus M. , de Haas Gerard H.
Published: December 2004

The role of aspartic acid‐49 (Asp‐49) in the active site of porcine pancreatic phospholipase A2 was studied by recombinant DNA techniques: two mutant proteins were constructed containing either glutamic acid (Glu) or lysine (Lys) at position 49. Enzymatic characterization indicated that the presence of Asp‐49 is essential for effective hydrolysis of phospholipids. Conversion of Asp‐49 to either Glu or Lys strongly reduces the binding of Ca2+ ions, in particular for the lysine mutant, but the affinity for substrate analogues is hardly affected. Extensive purification of naturally occurring Lys‐49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein that was nearly inactive. Inhibition studies showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys‐49 homologue itself has no enzymatic activity. Our results indicate that Asp‐49 is essential for the catalytic action of phospholipase A2. The importance of Asp‐49 was further evaluated by comparison of the primary sequences of 53 phospholipases A2 and phospholipase homologues showing that substitutions at position 49 are accompanied by structural variations of otherwise conserved residues. The occurrence of several nonconserved substitutions appeared to be a general characteristic of nonactive phospholipase A2 homologues.
Transforming growth factor‐β does not alter interleukin‐1 expression in cultured human macrophages
Authors: Hurme Mikko , Sihvola Marja , Alitalo Kari , Keski‐Oja Jorma
Published: December 2004

Transforming growth factor‐β (TGFβ) is a growth modulator that stimulates the growth of fibroblastic cells but inhibits the growth of cells of epithelial origin. TGFβ also influences the production of extracellular matrix proteins, and of proteases and the type 1 plasminogen activator inhibitor (PAI‐1) by cultured cells. TGFβ appears also to have various immunoregulatory effects, suppressing both T‐ and B‐cell activities. It has been proposed that it might increase the expression of interleukin‐1 (IL‐1) mRNA in cultured human monocytes, thus potentiating immune functions. To analyze the role of TGFβ in IL‐1 production we have now quantitated the effect of this factor on the production of biologically active IL‐1 as well as IL‐1β mRNA expression. The effect of TGFβ on IL‐1 production optimally activated with bacterial lipopolysaccharide (LPS) was also studied. It was found that IL‐1 activity and mRNA levels were rapidly elevated by LPS but not by TGFβ. Culture fluids from monocytes treated with TGFβ alone or with TGFβ plus LPS inhibited the proliferation of the test thymocytes. After gel filtration, the media from TGFβ‐treated cultures showed no activity in the molecular weight area of IL‐1 (approx. 15 kD), while the supernatants from TGFβ plus LPS‐induced cells contained IL‐1 activity in these fractions, the magnitude of which was, however, at the same level as in the culture fluids derived from cells stimulated with LPS alone. Thus our results show that the TGFβ used was biologically active but they provide no evidence for TGFβ in the regulation of IL‐1 production in human monocytes.
Transforming growth factor beta 1 (TGF‐β1) receptor expression on resting and mitogen‐activated T cells
Authors: Ellingsworth Larry , Nakayama Debra , Dasch James , Segarini Patricia , Carrillo Pedro , Waegell Wendy
Published: December 2004

Transforming growth factor β1 (TGF‐β1) is a potent autocrine growth inhibitor of lymphocytes. In this study, the expression of TGF‐β1 binding proteins was characterized on murine splenic T cells. With an affinity cross‐linking method and by neutralizing antibodies to TGF‐β1, [125I] TGF‐β1 was found to bind to three cell surface–binding proteins (280–200 kD, 95–85 kD, 65 kD) that were differentially expressed on resting and mitogen‐stimulated T cells. Freshly prepared (resting) T cells were found to constitutively express the 95–85‐kD form of these binding proteins, whereas mitogenic stimulation by either concanavalin‐A (Con‐A), interleukin‐1 (IL‐1), interleukin‐2 (IL‐2), or 12‐tetradencanoyl‐phorbol‐13‐acetate (TPA) for 12–72 h induced the appearance of all forms of the TGF‐β1 binding proteins (280–200 kD, 95–85 kD, and 65 kD). Furthermore, antibodies that neutralized the biologic action of TGF‐β1 also blocked the binding of [125I] TGF‐β1 to all three binding proteins, suggesting that these binding proteins are involved with signal transduction. These results suggest that the expression of the TGF‐β1 receptor on T cells is regulated by T cell mitogenic signals and that a regulatory relationship may exist between T cell growth‐promoting cytokines (IL‐1 and IL‐2) and the T cell growth inhibitor, TGF‐β1.
Molecular and cellular analysis of histamine H1 receptors on cultured smooth muscle cells
Authors: Mitsuhashi Masato , Payan Donald G.
Published: December 2004

Histamine is an important mediator of immediate hypersensitivity for both animals and humans. The action of histamine on target tissues is believed to be mediated by specific cell surface receptors, especially H1 and H2 receptors for hypersensitivity and inflammatory reactions, which involve stimulation of smooth muscle contractility, alterations in vascular permeability, and modifications in the activities of macrophages and lymphocytes. Although the nature of histamine receptors in the brain and peripheral tissues has been studied extensively by many laboratories, the molecular mechanism of histamine receptor‐mediated reactions is not fully understood, mainly because histamine receptors are incompletely characterized from the biochemical point of view. In previous studies, we have found that the cultured smooth muscle cell line DDT1MF‐2, derived from hamster vas deferens, expresses low‐affinity histamine H1 receptors and responds biochemically and functionally to H1‐specific stimulation (Mitsuhashi and Payan, J Cell Physiol 134:367, 1988). This cell line provides a model for analyzing the biochemical responses of H1 receptor‐mediated reactions in peripheral tissues. In this review, we summarized our recent progress in the study of low‐affinity H1 receptors on DDT1MF‐2 cells.
Competitive, uncompetitive, and mixed inhibitors of the alkaline phosphatase activity associated with the isolated brush border membrane of the tapeworm Hymenolepis diminuta
Authors: Pappas Peter W. , Leiby David A.
Published: December 2004

Several compounds were tested as inhibitors of the alkaline phosphatase (AlkPase) activity associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta. Molybdate, arsenate, arsenite and β‐glycerophosphate (BGP) were competitive inhibitors of the hydrolysis of p‐nitrophenyl phosphate, while levamisole and clorsulon were uncompetitive and mixed inhibitors, respectively. Molybdate was also a competitive inhibitor of the hydrolysis of BGP and 5′‐adenosine monophosphate, and levamisole was an uncompetitive inhibitor of BGP hydrolysis. The apparent inhibitor constants (K′i) for molybdate and levamisole were virtually identical regardless of the substrate, and these data support the hypothesis that the AlkPase activity is represented by a single membrane‐bound enzyme with low substrate specificity. Quinacrine, Hg2+, and ethylenediaminetetraacetate were also potent inhibitors of the AlkPase activity, but the mechanisms by which these latter three inhibitors function were not clear.
Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic‐AMP‐dependent protein kinases
Authors: Dawson Glyn , McAtee Patrick
Published: December 2004

Myelin basic protein, an 80‐kilodalton (kDa) protein in rat oligodendrocytes, and an 80‐kDa basic protein in neuroblastoma × neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl‐acetyl‐glycerol). TPA‐stimulated phosphorylation was inhibited by pre‐incubation with 50 μM psychosine (galactosyl‐sphingosine), confirming that it is mediated through the phospholipid‐dependent protein kinase C (PK‐C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic‐AMP‐dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte‐specific 2′,3′‐cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK‐C activation is discussed.
Coupling of antagonistic signalling pathways in modulation of neutrophil function
Authors: Mueller Heinz , Sklar Larry A.
Published: December 2004

Modulation of neutrophil activation by catecholamines reflects a fine‐tuning by coupling inhibitory and stimulatory receptor pathways. The catecholamine isoproterenol (ISO) binds to beta‐adrenergic cell surface receptors and thereby inhibits cell responses such as O production stimulated by formyl peptides. However, ISO did not inhibit O generation activated by 1 μM ionophore A23187, the protein kinase C activators phorbol ester (PMA, 100 ng/ml) and oleoylacetylglycerol (OAG, 50 μM), and the G‐protein activator NaF (40 mM). Furthermore, the overall kinetics of oxidant production in the presence of ISO were unchanged when cells were stimulated with PMA, OAG, A23187, and NaF. These results would imply that neither intracellular calcium, the activation of protein kinase C, nor the activation of G‐protein are the primary target of the inhibitory pathway. Accordingly, pertussis toxin did not block PMA or NaF‐stimulated superoxide generation. In contrast, formyl peptide‐dependent GTPase activity is inhibited by ISO in sonicated cell preparations. Since ISO increases the cAMP concentration in the cell, the possibility is raised that a cAMP‐dependent kinase inhibits signal transduction in part by blocking the interaction of this receptor with its G‐protein.
Design and synthesis of conformationally restricted phospholipids as phospholipase A2 inhibitors
Authors: Magolda R. L. , Galbraith W.
Published: December 2004

The use of conformationally restricted phospholipids 1 and 2 has been employed to understand the conformational preference of phospholipase A2 (PLA2) for substrate phospholipids. Inhibition of porcine pancreatic PLA2 with 1 and 2 indicated a two‐ to fivefold preference for the distal isomer 2 over the proximal isomer 1. Based upon these studies, both side‐chains of the substrate phospholipid appear to occupy the lipid binding domains near the active site with the side‐chains further apart most preferred by PLA2.
Differential effect of dexamethasone on the regulation of phospholipase A2 and prostanoid synthesis in undifferentiated and phorbolester‐differentiated U937 cells
Authors: Koehler L. , Hass R. , Goppelt‐Struebe M. , Kaever V. , Resch K.
Published: December 2004

The human undifferentiated histiocytic cell‐line U937 can be induced to differentiate by incubation with 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) into macrophage‐like cells. Dexamethasone reduced the prostaglandin production in TPA‐differentiated U937 cells dose dependently, whereas undifferentiated U937 cells were dexamethasone insensitive. Concomitantly phospholipase A2, the enzyme liberating the prostaglandin precursor arachidonic acid, was inhibited by dexamethasone in TPA‐differentiated but not in undifferentiated U937 cells. The activity of lysophosphatide acyltransferase, the key enzyme of fatty acid reacylation into phospholipids, remained unchanged both in undifferentiated and TPA‐differentiated U937 cells. The data suggest that responsiveness to glucocorticoid‐dependent regulation of prostanoid synthesis is acquired by cells of the monocyte‐macrophage lineage late in differentiation.
Altered actin and immunoglobulin Cμ expression in nitrogen mustard‐resistant human Burkitt lymphoma cells
Authors: Tan K. B. , Grillone Lisa , Boyce Rebecca , Crooke Stanley T.
Published: December 2004

Raji‐HN2 is a B cell lymphoma (Burkitt lymphoma) line that was made resistant to nitrogen mustard. The drug‐resistant phenotype was accompanied by changes in gene expression. The expression of four unrelated genes was examined by Northern blot analysis. Raji‐HN2 cells were found to contain about twice the number of actin mRNA found in Raji cells. Both cell lines were found to contain equivalent amounts of β2‐microglobulin, c‐myc oncogene, and immunoglobulin Cμ mRNAs. The Cμ mRNA was, however, larger in size in Raji‐HN2 cells. Alterations in actin and Cμ mRNAs in Raji‐HN2 cells were not due to gene amplification or rearrangement because Southern blot analysis revealed no changes in the genomic organization of these genes. The increased actin mRNA content was correlated with an increased actin content of Raji‐HN2 cells. The F‐actin (stained with 7‐nitrobenz‐2‐oxa‐1,3‐diazolylphallacidin) content of single cells was quantitated in a meridian interactive laser cytometer. Raji‐HN2 cells contained about twice the amount of F‐actin present in the parental Raji cells. Similar results were obtained when large populations, 106 cells each, were examined in a flow cytometer.
Effects of bombesin on human small cell lung cancer cells: Evidence for a subset of bombesin non‐responsive cell lines
Authors: Kado‐Fong Helen , Malfroy Bernard
Published: December 2004

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI‐H69, NCI‐H128, and NCI‐H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro‐gastrin releasing peptide (GRP), only the NCI‐H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I‐GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.
Culture of primary lung tumors using medium conditioned by a lung carcinoma cell line
Authors: Siegfried Jill M.
Published: December 2004

Medium conditioned by the established lung tumor cell line A549 was used as a supplement to culture cells from primary solid lung tumors. Of 36 cases placed into culture, primary cells were obtained in 33 (91.7%). Of 29 cases in which subcultures were attempted, 18 (62.1%) were successful. Nine cell lines have been established by this technique to date. In growth assays, conditioned medium (CM) was found to stimulate both monolayer colony formation and growth in semi‐solid medium of cells cultured from primary solid tumors. CM has been found to contain factors with the properties of both transforming growth factorα (TGFα) and insulin‐like growth factor‐I (IGF‐I). The addition of a combination of these factors as purified peptides to basal medium at levels found in CM (0.1–0.5 ng/ml) stimulated colony formation of lung tumor cells by up to fourfold. These results indicate that secretion of growth factors may be important in tumor growth in vivo, and that use of CM may be a valuable tool for obtaining cultures from primary solid tumors.