bluuebunny/crossref_metadata_2025 huggingface dataset:
Affinity isolation of active murine erythroleukemia cell chromatin: Uniform distribution of ubiquitinated histone H2A between active and inactive fractions
Authors: Dawson Barbara A. , Herman Tim , Haas Arthur L. , Lough John
Published: May 2005

This laboratory recently reported the development of a biotin‐cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15‐fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830–12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta‐globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5‐[(N‐biotin‐amido)hexanoamido‐ethyl‐1,37apos;‐dithiopropionyl‐3‐aminoallyl]‐2' ‐deoxyuridine 5'‐triphosphate (Bio‐19‐SS‐dUTP), during a nick‐translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non‐biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one‐half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta‐globin sequences were associated with chromatin fragements which had bound to the affinity complex. Western blot assessment for ubiquitinated histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.
Fluorescent tetradecanoylphorbol acetate: A novel probe of phorbol ester binding domains
Authors: Balázs Margit , Szöllösi Jánòs , Lee William C. , Haugland Richard P. , Guzikowski Anthony P. , Fulwyler Mack J. , Damjanovich Sandor , Feuerstein Burt G. , Pershadsingh Harrihar A.
Published: May 2005

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol‐13‐acetate‐12‐N‐methyl‐N‐4‐(N,N′‐di(2‐hydroxyethyl)amino)‐7‐nitrobenz‐2‐oxa‐1,3‐diazole‐aminododecanoate (N‐C12‐Ac(13)) of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N‐C12‐Ac(13) is close to 488 nm, facilitating its use in argon‐ion laser flow and imaging cytometry. When incubated with 100 nM N‐C12‐Ac(13) at 25°C, P3HR‐1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20‐fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N‐C12‐Ac(13) stained cells in a dose‐dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]‐phorbol‐12,13‐dibutyrate ([3H]‐PBu2) from rat brain cytosol with N‐C12‐Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N‐C12‐Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N‐C12‐Ac(13), at a lower concentration (51 nM), induced expression of Epstein‐Barr viral glycoprotein in P3HR‐1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N‐C12‐Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non‐specific binding of N‐C12‐Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non‐specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.
Quantitative variations in gene expression: Possible role in cellular diversification and tumor progression
Authors: Nicolson Garth L.
Published: May 2005

Changes in the quantitative expression of certain genes or in the amounts of their products can quickly stimulate progression to the metastatic phenotype. This has been done experimentally by transferring dominantly acting oncogenes such as c‐H‐rasEJ into susceptible cells or more recently by interfering with metastasis suppressor genes. In vivo such rapid qualitative changes in dominantly acting oncogenes or suppressor genes occur only rarely, and progression to highly metastatic phenotypes is thought to occur through a process involving the slow stepwise progression of a subpopulation of neoplastic cells to more malignant states. Such slow changes can be reversible and need not involve known dominantly acting oncogenes or metastatic suppressor genes, consistent with clinical and experimental observations on naturally occuring, highly advanced metastatic tumors. An important element in the natural progression of tumors to more malignant states may be their ability to circumvent host environmental controls that regulate growth and cellular diversity. They also evolve into heterogeneous cellular phenotypes, a process that appears to mainly involve quantitative changes in gene expression but can be rapidly stimulated in cell culture by the introduction of a dominantly acting oncogene or inhibited by the introduction of a suppressor gene. The oncogenes and suppressor genes that affect malignancy may control important steps in the quantitative regulation of sets of genes that are ultimately responsible for the cellular alterations seen in adhesion receptors, cell motility responses, cell‐cell communication components, degradative enzymes and their inhibitors, growth factor receptors, components that aid in escape from host surveillance mechanisms and others that are important in malignancy. Highly malignant cells that have slowly evolved in vivo may contain only a few qualitative gene changes but have undergone extensive cycles of diversification and accumulation of quantitative changes in the expression of genes that encode products that are related to malignancy and metastasis. Thus highly malignant cells can arise quickly due to specific qualitative changes in critical controlling genes or more slowly by less critical qualitative genetic changes together with cycles of cellular diversification and accumulation of quantitative changes in gene expression.
Association of vanadate‐sensitive Mg2+‐ATPase and shape change in intact red blood cells
Authors: Xu Y.‐H. , Lu Z.‐Y. , Conigrave A. D. , Auland M. E. , Roufogalis B. D.
Published: May 2005

Intact human erythrocytes, initially depleted of Mg2+ by EDTA incubation in the presence of A23187, exhibit Mg2+‐dependent phosphate production of around 1.5 mmol per liter cells · h, half‐maximally activated at around 0.4 mM added free Mg2+. This appears to correspond to Mg2+‐stimulated adenosine triphosphatase (Mg2+‐ATPase) activity found in isolated membranes, which is known to have a similar activity and affinity for Mg2+. Vanadate (up to 100 μM) inhibited Mg2+‐dependent phosphate production and ATP breakdown in intact cells. Over a similar concentration range vanadate (3–100 μM) transformed intact cells from normal discocytes to echinocytes within 4–8 h at 37°C, and more rapidly in Mg2+‐depleted cells. The rate of Ca2+‐induced echinocytosis was also enhanced in Mg2+‐depleted cells. These results support previous studies in erythrocyte ghosts suggesting that vanadate‐induced shape change is associated with inhibition of Mg2+‐ATPase activity localized in the plasma membrane of the red blood cell.
Facile platelet adhesion to collagen requires metabolic energy and actin polymerization and evokes intracellular free calcium mobilization
Authors: Smith J. Bryan , Dangelmaier Carol , Selak Mary A. , Daniel James L.
Published: May 2005

The attachment of platelets to collagen‐coated microtiter plates at 20°C was inhibited strongly by depletion of metabolic energy or by addition of cytochalasins and was slightly inhibited by the intracellular Ca2+ chelator BAPTA. In keeping with their respective potencies as inhibitors of actin polymerization, cytochalasins D and H were the most potent inhibitors of adhesion, while cytochalasin B was the least potent. Energy depletion, cytochalasin D or, to a much lesser extent, BAPTA also inhibited platelet adhesion to collagen in a suspension assay system at 37°C. Collagen‐induced platelet cytosolic Ca2+ mobilization was inhibited up to 70% by cytochalasin D and abolished by energy depletion or BAPTA. Elevation of intracellular platelet calcium by treatment with ionomycin had little effect on platelet adhesion to collagen. We propose that rapid platelet spreading along collagen fibers is both energy‐ and actin‐dependent and necessary to produce maximal adhesion needed to elicit Ca2+ mobilization required for subsequent responses.
Tumorigenesis in transgenic mice: Identification and characterization of synergizing oncogenes
Authors: Berns Anton
Published: May 2005

Transgenic mice carrying oncogenes present a useful model with which to assess the tissue‐specific action of oncogenes. These mice are usually predisposed to a specific type of neoplastic growth. The tumors that arise are usually monoclonal in origin and become only apparent after a variable latency period, suggesting that additional events are required for tumor formation. Identification of these additional events is highly relevant: it might give access to the genes that can synergize with a preselected oncogene in tumorigenesis and could facilitate the identification of the biochemical pathways in which these genes act. Retroviruses can be instrumental in identifying cooperating oncogenes. Proto‐oncogene activation or tumor suppressor gene inactivation by insertional mutagenesis is an important mechanism by which the non‐acute transforming retroviruses can induce tumors in several species. Owing to the sequence tag provided by the provirus, the relevant proto‐oncogene can be directly identified by cloning of the DNA flanking the proviral insertion site. We have exploited this potential of retroviruses by infecting Eμ‐pim‐1 and Eμ‐myc transgenic mice, which are predisposed to lymphomagenesis, with Moloney murine leukemia virus (MuLV). A strong acceleration of tumor induction ensued upon infection of these mice with MuLV. More importantly, it allowed us to identify a number of additional common insertion sites marking both previously known as well as new (putative) oncogenes. In a significant portion of the tumors more than one oncogene was found to be activated, indicating that within this system the synergistic effect of at least three genes can be established. Furthermore, the distribution of proviruses over these common insertion sites suggests that this methodology enables us to assign each of these genes to distinct complementation groups in tumorigenesis. To get insight into the function of these oncogenes, we have inactivated one of them, the pim‐1 oncogene, via homologous recombination in ES cells. We have shown that by using a pim‐1 targeting vector in which transcription and translation of the resistance marker was made dependent on the acquisition of both a promoter and an in‐frame translational initiation codon, the gene could be inactivated with high efficiency. Subsequently, we inactivated the second allele by using a different selectable marker. The absence of the pim‐1 gene product does not appear to have any detrimental effect on the growth of ES cells and hematopoietic cells derived from these ES cells in vitro, despite the fact that pim‐1 is highly expressed in wild‐type ES cells. More detailed analysis of pim‐1‐less cells and mice will be necessary to gain insight into the relevance of the highly conserved pim‐1 proto‐oncogene in development and maintenance of mammalian organisms.
Platelet‐derived endothelial cell growth factor
Authors: Heldin Carl‐Henrik , Usuki Kensuke , Miyazono Kohei
Published: May 2005

Platelet‐derived endothelial cell growth factor (PD‐ECGF) is a 45kDa single chain polypeptide which stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo. Analysis of a full length PD‐ECGF cDNA revealed an open reading frame coding for 482 amino acids without homology to other known proteins. No signal sequence was observed, and analysis of the biosynthesis and processing of PD‐ECGF in a thyroid carcinoma cell line revealed that PD‐ECGF is released only very slowly. PD‐ECGF becomes covalently associated with nucleotide triphosphates (e.g., ATP) in vivo, as well as in vitro. The physiological significance of this posttranslational modification remains to be elucidated. The tissue distribution and target cell specificity of PD‐ECGF suggests roles in angiogenesis (e.g., during wound healing and in the developing placenta), as well as in the maintenance of the integrity of the endothelial cell lining of large vessels.
The vascular endothelial growth factor family of polypeptides
Authors: Ferrara Napoleone , Houck Keith A. , Jakeman Lyn B. , Winer Jane , Leung David W.
Published: May 2005

Vascular endothelial growth factor (VEGF) was identified as a heparin‐binding polypeptide mitogen with a target cell specificity restricted to vascular endothelial cells. Molecular cloning reveals the existence of four species of VEGF having 121, 165, 189, and 206 amino acids. These have strikingly different secretion patterns, which suggests multiple physiological roles for this family of polypeptides. The two shorter forms are efficiently secreted, while the longer ones are mostly cell‐associated. Alternative splicing of mRNA, rather that transcription from different genes, is the mechanism for their generation. In situ hybridization reveals that the VEGF mRNA is widely distributed in most tissues and organs and expressed at particularly high levels in areas of active vascular proliferation, like the ovarian corpus luteum. Ligand autoradiography on rat tissue sections demonstrates that VEGF binding sites are associated with vascular endothelial cells of both fenestrated and non‐fenestrated capillaries and with the endothelium of large vessels, while no displaceable binding is evident on non‐endothelial cell types. These findings support the hypothesis that VEGF plays a highly specific role in the maintenance and in the induction of growth of vascular endothelial cells.
Endothelial cell regulation by transforming growth factor‐beta
Authors: RayChaudhury Amlan , D'Amore Patricia A.
Published: May 2005

Pronounced changes including growth inhibition, increased matrix deposition and suppression of cell‐associated proteolytic activity, take place in endothelial cells (EC) upon the application of TGF‐β. Interrelationships between these effects have shed some light on the mechanism of action of TGF‐β and on its role in regulating EC function vis‐a‐vis angiogenesis. For instance, preliminary evidence has indicated that increased levels of certain matrix components may be partly responsible for the antiproliferative action of TGF‐β. In addition, TGF‐β and bFGF have opposing effects on cellular proteolytic balance which may contribute to the antagonistic effect that TGF‐β has on bFGF‐induced EC growth and possibly to the anti‐angiogenic effect exerted by TGF‐β under certain circumstances. Of particular interest in this regard is the fact that physical contact between EC and vascular mural cells in EC:mural cell cocultures has been found to generate active TGF‐β, thus further implicating TGF‐β in the maintenance of the quiescent, differentiated aggreation of EC as found in vascular structures in vivo. While more information is needed to define what, if any, role TGF‐β plays in endothelial differentiation, it is to be noted that many of the cellular and biochemical processes affected by TGF‐β are linked to differentiation. It is therefore possible that the growth inhibition of EC by TGF‐β primes them for differentiation and/or is critical for the maintenance of a differentiated state.
Substrates and signalling complexes: The tortured path to insulin action
Authors: Roth Richard A. , Zhang Bei , Chin Janice E. , Kovacina Kristina
Published: May 2005

In the last few years several potential substrates of the insulin receptor tyrosine kinase have been identified, purified, and their cDNAs isolated. These putative substrates include: (1) pp15, a fatty acid‐binding protein; (2) pp120, a plasma membrane ecto‐ATPase; (3) pp42, a MAP serine/threonine kinase; (4) pp85, a subunit of the Type 1 phosphatidylinositol kinase; and (5) pp185, a phosphatidylinositol kinase binding protein. Although the tyrosine phosphorylation of several of these substrates correlates with the signalling capabilities of various mutant receptors, the role of these substrates in mediating any one of insulin's many biological responses is still unknown. In addition, recent data indicate that the tyrosine phosphorylation of pp42 may in fact be due to autophosphorylation, thereby removing it from the list of putative substrates of the insulin receptor kinase. Finally, the present review discusses the question of whether signalling occurs as a result of the tyrosine phosphorylation of substrates or via the formation of signalling complexes.
Modulation of collagen and fibronectin synthesis in fibroblasts by normal and malignant cells
Authors: Noel A. , Lambert C. A. , Nusgens B. , Lapiere C. M. , Munaut C. , Foidart J. M. , Boulvain A. , Calberg‐Bacq C. M.
Published: May 2005

The influence of various normal and malignant human cells on the level of collagen synthesis by human fibroblasts was tested in coculture. As revealed by immunoperoxidase staining, in cocultures with breast adenocarcinoma cells (MCF7, SA52, T47D) fibroblasts synthesized collagen while tumor cells did not. Fibroblasts displayed increased collagen production without change in the overall protein synthesis. Several other types of cells derived from normal human tissues (keratinocytes, normal mammary cells) or from fibrosarcoma, melanoma, cervical carcinoma, choriocarcinoma, or other breast adenocarcinoma (SW613, MDA, BT20) did not affect collagen synthesis of fibroblasts. Although to a lesser extent, this stimulating effect was reproduced by using the conditioned medium (CM) of the active cells but not with CM of the other cell types. A slight stimulation was also obtained when tumoral MCF7 cells and fibroblasts shared the same medium but were physically separated, suggesting that close contact was required for optimal stimulation of collagen synthesis. The collagen synthesis stimulating activity was not related to a modification of fibroblast proliferation rate. The production of collagen types I, III, and VI and fibronectin were increased in cocultures of fibroblasts with MCF7 cells. The increased synthesis of collagen types I and III and fibronectin was paralleled by similar changes in the steady‐state level of their mRNAs. On the contrary, the increased production of collagen type VI appeared regulated at a post‐transcriptional level.
Comparison between avian and human prolyl 4‐hydroxylases: Studies on the holomeric enzymes and their constituent subunits
Authors: Guzman Norberto A. , Ascari William Q. , Cutroneo Kenneth R. , Desnick Robert J.
Published: May 2005

Prolyl 4‐hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans‐hydroxyproline in nascent or completed pro‐α chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated α and β. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4‐hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4‐hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4‐hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high‐resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins.

Electrophoretic studies revealed that the human prolyl 4‐hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated α and β, respectively. In contrast, the chick embryo α and β subunit ratio was 1 to 1. Notably, the human α subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the α subunit was observed. Finally, only the α subunit was bound to Concanavalin A demonstrating that the α subunits of prolyl 4‐hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4‐trans‐hydroxy‐L‐proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well‐characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.
Expression of heat shock genes during differentiation of mammalian osteoblasts and promyelocytic leukemia cells
Authors: Shakoori Abdul Rauf , Oberdorf Annette M. , Owen Thomas A. , Weber Lee A. , Hickey Eileen , Stein Janet L. , Lian Jane B. , Stein Gary S.
Published: May 2005

The progressive differentiation of both normal rat osteoblasts and HL‐60 promyelocytic leukemia cells involves the sequential expression of specific genes encoding proteins that are characteristic of their respective developing cellular phenotypes. In addition to the selective expression of various phenotype marker genes, several members of the heat shock gene family exhibit differential expression throughout the developmental sequence of these two cell types. As determined by steady state mRNA levels, in both osteoblasts and HL‐60 cells expression of hsp27, hsp60, hsp70, hsp89α, and hsp89β may be associated with the modifications in gene expression and cellular architecture that occur during differentiation.

In both differentiation systems, the expression of hsp27 mRNA shows a 2.5‐fold increase with the down‐regulation of proliferation while hsp60 mRNA levels are maximal during active proliferation and subsequently decline post‐proliferatively. mRNA expression of two members of the hsp90 family decreases with the shutdown of proliferation, with a parallel relationship between hsp89α mRNA levels and proliferation in osteoblasts and a delay in down‐regulation of hsp89α mRNA levels in HL‐60 cells and of hsp89β mRNA in both systems. Hsp70 mRNA rapidly increases, almost twofold, as proliferation decreases in HL‐60 cells but during osteoblast growth and differentiation was only minimally detectable and showed no significant changes. Although the presence of the various hsp mRNA species is maintained at some level throughout the developmental sequence of both osteoblasts and HL‐60 cells, changes in the extent to which the heat shock genes are expressed occur primarily in association with the decline of proliferative activity. The observed differences in patterns of expression for the various heat shock genes are consistent with involvement in mediating a series of regulatory events functionally related to the control of both cell growth and differentiation.
Biochemical and morphological differentiation of the human colonic epithelial cell line SW620 in the presence of dimethylsulfoxide
Authors: Omary M. Bishr , de Grandpre Louise , McCaffrey Michael , Kagnoff Martin F.
Published: May 2005

In vitro models of intestinal cell differentiation provide an important adjunct for studying normal and abnormal intestinal epithelial cell diffrentiation. The studies reported herein describe morphologic and biochemical changes in the colonic epithelial cell line SW620 following dimethylsulfoxide (DMSO) incubation. Cells cultured in the presence of DMSO showed striking changes in morphology characterized by enlargement, elongation, and formation of process‐like structures by light microscopy and a propensity to form microvillus‐like structures by electron microscopy. These changes were accompanied by significant differences in the expression of the cell surface markers CD4 (HIV gp120 receptor), CD44 (hyaluronate receptor), and KS1 (adenocarcinoma/epithelial specific antigen). There was a marked decrease in CD4 expression (38% to 2%), an increase in CD44 expression (4% to 50%) and a decrease in KS1 expression (98% to 66%) as detected by flow cytometry following incubation of SW620 cells in DMSO. Parallel changes in the expression of these markers were seen by metabolic and surface labeling studies. Although SW620 cells were infected by HIV‐1, DMSO‐treated SW620 cells could not be infected. DMSO‐induced changes in surface expression of CD4, CD44, and KS‐1 were reversible over time upon removal of DMSO from the culture medium. Secretory component, sucrase, neuron‐specific enolase, chromogranin‐A and mucin were not detectable in SW620 cells with or without DMSO treatment. SW620 cells provide a useful model for studying specific biochemical and molecular events involved in intestinal epithelial cell differentiation and function.
The inhibition of ATP‐dependent shape change of human erythrocyte ghosts correlates with an inhibition of Mg2+‐ATPase activity by fluoride and aluminofluoride complexes
Authors: Morris Michael B. , Monteith Gregory , Roufogalis Basil D.
Published: May 2005

The vanadate‐sensitive Mg2+‐dependent ATPase activity of the human erythrocyte ghost is believed to be involved in the shape change events that convert echinocytic ghosts to smoothed forms (biconcave discs and stomatocytes). At physiological salt concentration, pH 7.4, 2 mMATP, 5 mM Mg2+ and 1 mM EGTA, the Mg2+‐ATPase activity of ghosts was inhibited strongly by millimolar concentrations of sodium fluoride: I50 = 1.31 ± 0.2, mM (mean ± S.D., n = 12). The addition of aluminium chloride to 15 μM reduced the concentration of NaF required for 50% inhibition to 0.76 ± 0.21 mM (n = 10). Aluminium alone had only a small inhibitory effect of the ATPase activity (13 ± 9 %; n = 10). Desferrioxamine, a strong chalator of tervalent aluminium ion, failed to reverse the inbibition by fluoride and reversed the inhibition in the presence of aluminium and fluoride back to those values obtained with fluoride alone. Of several metal salts tested only beryllium sulfate was able to replace aluminium as an effective inhibitor in the presence of fluoride.

Inhibition of the Mg2+‐ATPase activity by fluoride and the aluminofluoride complexes correlated with an inhibition of the rate of MgATP‐dependent change in red cell ghost shape from echinocytes to smoothed forms. All gross morphological changes of the smoothing process were affected, including the production of discocytes, stomatocytes and endocyctic vesicles.
Growth regulation of the mammalian ocular lens by vitreous humor
Authors: Banerjee Amit , Parafina Julian , Bagchi Mihir
Published: May 2005

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95°C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor‐receptor binding is essential for the growth inhibitory effect.
Wild‐type murine p53 represses transcription from the murine c‐myc promoter in a human glial cell line
Authors: Moberg Kenneth H. , Tyndall W. Andrew , Hall David J.
Published: May 2005

Here we analyzed the effect of the suppressor proto‐oncogene p53 on transcription from the P2 promoter of the murien c‐myc gene. c‐myc promoter constructs were coupled to the chloramphenical acetyl‐transferase (CAT) gene and were transiently transfected into a human glial cell line along with plasmids overexpressing wild‐type or mutant p53. It was found that significant repression of c‐myc transcription took place following cotransfection with wild‐type but not mutant p53. However wild‐type p53 did not suppress transcription from the SV40 early promoter or from the MHC promoter. Promoter‐CAT constructs containing only the ME1a2 or E2F elements, from the P2 promoter, were repressed by p53, indicating that p53 may exert its effect at these two sites within the P2 promoter. Finally, when the SV40 T antigen and wild‐type p53 were expressed together in glial cells the repressive effect of p53 was abolished.
Identification of a fat cell enhancer: Analysis of requirements for adipose tissue‐specific gene expression
Authors: Graves Reed A. , Tontonoz Peter , Platt Kenneth A. , Ross Susan R. , Spiegelman Bruce M.
Published: May 2005

The molecular basis for adipose‐specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5′‐flanking region of the adipocyte P2 (aP2) gene to direct cell‐type specific gene expression. Although the proximal promoter containing AP‐1 and C/EBP binding sites is capable of directing differentiation‐dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that −5.4 kb of the 5‐flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at −5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation‐dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis‐ and transacting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.
Embryonic stem cells and in vitro hematopoiesis
Authors: Snodgrass H. Ralph , Schmitt Regina M. , Bruyns Eddy
Published: May 2005

To study hematopoietic differentiation a variety of in vitro systems have been established using hematopoietic precursors derived from various explanted adult and fetal tissues. In this prospective we describe and discuss the potential of a novel system for studying the earliest stages of hematopoietic development. In addition, some of the applications of this system as a unique in vitro model for studying other developmental systems are discussed. Murine embryonic stem cells (ESC), which are totipotent and can be maintained undifferentiated indefinitely in vitro, have the capacity to differentiate in vitro into hematopoietic precursors of most, if not all, of the colony forming cells found in normal bone marrow. This potential can be exploited to study the control of the early stages of hematopoietic induction and differentiation. Recent results have indicated that there is a strong transcriptional activation, in a well defined temporal order, of many of the hematopoietically relevant genes. Examples of the genes expressed early during the induction of hematopoiesis include erythropoietin (Epo) and its receptor as well as the Steel (SI) factor (SLF) and its receptor (c‐kit). Several other genes, including CSF‐1, IL‐1, and G‐CSF were expressed during the later stages of hematopoietic differentiation. Contrasting with these observations, IL‐3 and GM‐CSF were not expressed during the first 24 days of ES cell differentiation suggesting that neither factor is necessary for the induction of hematopoietic precursors. Although these studies are just beginning, this system is easily manipulated and gives us an approach to understanding the control of the induction and differentiation of the hematopoietic system in ways not previously possible.
Expression of glucose transporters (GLUT 1 and GLUT 4) in primary cultured rat adipocytes: Differential evolution with time and chronic insulin effect
Authors: Hajduch Eric J. , Guerre‐Millo Michèle C. , Hainault Isabelle A. , Guichard Colette M. , Lavau Marcelle M.
Published: May 2005

We previously reported that in cultured adipose cell lines insulin increased selectively the expression of Glut 1, in contrast to in vivo regulation where variations in insulinemia have been shown to affect only GLUT 4. We have addressed here the question of the long‐term regulation of GLUT 1 and GLUT 4 in fat cells by using primary cultures of rat adipocytes. Epididymal fat cells were isolated by collagenase and cultured 4 days in DMEM supplemented with BSA 1%, FCS 1%, and glucose 10 mM. GLUT 1 and GLUT 4 proteins were assessed in total cellular membranes by Western blotting, using specific antibodies against their respective C‐terminal peptides. GLUT 1 steadily increased over culture time to reach at day 3, a level 3‐fold higher than the initial value. In contrast, GLUT 4 decreased sharply and stabilized at day 3, at 30% of the initial value. The changes in GLUT 1 and GLUT 4 mRNAs with culture time were parallel to changes in the corresponding proteins, suggesting a pre‐translational level of regulation. The expression of the lipogenic enzyme, fatty acid synthetase (FAS), highly expressed in fat cell, decreased over time following a pattern closely parallel to that of GLUT 4. Chronic exposure to insulin added at day 2 had no effect on GLUT 4 expression but increased the expression of GLUT 1 and FAS by 70% and 36%, respectively. Glucose consumption was stable over 4 days of culture, while lactate production increased from 24 to 36% of glucose utilization, in agreement with the loss in FAS. Glucose consumption increased only slightly with insulin (160%), in good keeping with the low levels of expression of both GLUT 4 and FAS in these cultured cells. These data indicate that culture alters oppositely the expression of GLUT 1 and GLUT 4 in rat adipocytes and suggest that factor(s) other than insulin predominate in their regulation in vivo.
Prolyl endopeptidase purified from granulomatous inflammation in mice
Authors: Nozaki Yukinori , Sato Naoyoshi , Iida Toshihiro , Hara Kenji , Fukuyama Kimie , Epstein William L.
Published: May 2005

Activity of prolyl endopeptidase (EC 3.4.21.26) which hydrolyses the Pro7‐Phe8 bond in angiotensin II has been found to elevate in experimentally produced granulomatous inflammation in liver and skin. We purified the enzyme 1,536‐fold by 6 steps from murine hepatic granulomas. The purified enzyme has a molecular weight of 79 kDa and physiocochemical properties equivalent to those previously reported for prolyl endopeptidase purified from other sources. By HPLC analysis, the cleavage of Phe8‐Leu10 and Phe8 from angiotensin I and II, respectively, was detected and quantified. Monospecific IgG was prepared from serum of rabbits injected with purified enzyme. Concentration of the enzyme was immunohistochemically detected in cells which form granulomatous organization, but not in inflammatory cells surrounding the foci. The antibody, however, cross reacted with the enzyme in adjacent liver cells and weakly stained their cytoplasm. The findings indicate that this enzyme, in addition to angiotensin converting enzyme, may serve as a useful biochemical marker for granulomatous tissue reactions.
Modification of blood group A antigen expression in a pancreatic cancer cell line (PC‐1) by inhibitors of N‐glycan processing
Authors: Hirota Masahiko , Mogaki Masatoshi , Pour Parviz M. , Chaney William G.
Published: May 2005

Pancreatic adenocarcinomas induced in Syrian hamsters by treatment with N‐nitrosobis(2‐oxopropyl) amine express blood group A antigen, which is absent in normal pancreatic cells. On membrane glycoproteins purified from tumors, blood group A antigen has been found to be expressed on multiantennary Asn‐linked complex glycans. In this study, we investigated the effect of inhibitors of Asn‐glycan processing on blood group A antigen bearing glycan structures in a cell line (PC‐1) established from a primary induced pancreatic cancer. Expression of blood group A antigen on cells and in membrane preparations was blocked by treatment with 1‐deoxymannojirimycin, an inhibitor of mannosidase I, but was retained after treatment with swainsonine, an inhibitor of mannosidase II. However, swainsonine treatment altered the glycan structure associated with blood group A antigen from an endoglycosidase H resistant type to a sensitive type, indicating that the blood group A structure might shift from a complex type to a hybr‐id type glycan by this treatment. These results demonstrate that Asn‐linked glycans carry the major blood group A antigens in PC‐1 cells. © 1992 Wiley‐Liss, Inc.
Evidence for the presence of complex high‐molecular mass N‐linked oligosaccharides in intranuclear gylcoproteins from hela cells
Authors: Codogno Patrice , Bauvy Chantal , Sève Annie‐Pierre , Hubert Michèle , Ogier‐Denis Eric , Aubery Michèle , Hubert Jean
Published: May 2005

Nonhistone proteins were extracted in 0.4 M NaCl from membrane‐depleted nuclie of HeLa cells grown in the presence or the absence of [5,6‐3H]fucose. Control experiments strongly suggest that most extracted proteins were needed nuclear components. Several protiens, present in the 0.4 M NaCl nuclear extract, with Mr ranging from 35,000 to 115,000 were identified on Western bolts as fucosylated glycoproteins owing to their binding to the fucose‐specific lectin, Ulex eruopeus agglutinin l. Results of experiments involving mild alkaline treatment and peptide N‐glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N‐linked to the polypeptide backbone. Analysis of the N‐glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the High‐Mr oligoaccharides to endo‐β‐galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N‐acetyllactosamine units [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fuose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoprotiens. © 1992 Wiley‐Liss, Inc.
Interaction of MAR‐sequences with nuclear matrix proteins
Authors: Ivanchenko Maria , Avramova Zoya
Published: May 2005

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273–282, 1984; Cockerill and Garrard: Cell 44:273–282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.; EMBO J 8:3997–4006, 1989; lzaurralde et al.: J Mol Biol 210:573–585, 1989]. Here, applying a different approach, we were able to “visualize” some of the skeletal proteins recognizing and specifically binding MAR‐sequences. It is shown that the major matrix proteins are practically the same in both salt‐ and LIS‐extracted matrices. However, the relative MAR‐binding activity of the individual protein components may be different, depending on the method of matrix preparation.

The immunological approach applied here allowed us to identify some of the individual MAR‐binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR‐sequences as well. Evidence is presented that proteins recognized by the anti‐HMG antibodies also participate in Mar‐interactions. © 1992 Wiley‐Liss, Inc.
Fluorescence cytometry of microtubules and nuclear DNA during cell‐cycle and reverse‐transformation
Authors: Vergani L. , Gavazzo P. , Facci P. , Diaspro A. , Mascetti G. , Nicolini C. , Arena N. , Gaspa L.
Published: May 2005

Synchronized CHO‐K1 cells and their dibutyryl c‐AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules.

In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI‐stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen‐stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase.

In the early part of G1, the drastic alteration in anti‐β tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide.

The known 1–2 h lengthening of the G1 period after reverse‐transformation appears to correlate with a similar delay in the abrupt chromatin decondensation.

The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast‐like cells. © 1992 Wiley‐Liss, Inc.
Recombinant murine steel factor stimulates in vitro production of granulocyte–macrophage progenitor cells
Authors: Williams Douglas E. , Foxworthe Diane , Teepe Mark , Eisenman June , Lyman Stewart D , Anderson Dirk
Published: May 2005

The ability of murine Steel factor to promote the in vitro production of granulocyte‐macrophage progenitor cells (CFU‐GM) was examined in short‐term liquid cultures. Bone marrow from C57BL/6J or Sl/Sld mice was placed in culture for seven days with either Steel factor alone or in the presence of IL‐3. CFU‐GM responsive to GM‐CSF, IL‐3, and CSF‐1 were measured in the input population and again after 3 or 7 days in culture. Steel factor alone increased the number of all CUF‐GM types as early as 3 days after culture initiation, with further increases at day 7. This effect was protentiated by the addition of IL‐3. Production of CFU‐GM by C57BL/6J or Sl/Sld marrow was comparable except for enhanced production of CSF‐1 responsive progenitors by Sl/Sld marrow. A recombinant Sld protein was also shown to be equivalent to the wild‐type protein in its capacity to promote CFU‐GM production from normal bone marrow. © 1992 Wiley‐Liss, Inc.
Activin A‐induced diffrentiation in K562 cells is associated with a transient hypophosphorylation of RB protein and the concomitant block of cell cycle at G1 phase
Authors: Sehy David W. , Shao Li‐En , Tsai Wei‐Min , Yu John , Yu Alice L.
Published: May 2005

The human erythroleukemic cell line, K562, can be induced to differentiate by the addition of activin A, a newly purified protein belonging to the TGF‐β1 family. The present studies used flow cytometric cell cycle analysis, indirect immunofluorescence staining of the proliferating cell nuclear antigen (PCNA), and thymidine incorporation assay of cell proliferation to study the effects of activin A on the cell cycle during differentiation in K652 cells. Activin phase. The latter can be observed after approximately 24 hr of incubation with activin A and then disappears after this early stage of induction of differentiation. Cell cycle kinetics analysis using synchronized K562 cells also confirms that in the presence of activin A, K562 cells progress normally through various phases of cell, except that there is prolongation of the G1 phase between 10 to 24 hr of culture. Furthermore, this transient arrest in G1 is correlated with dephosphorylation of a nucleoprotein, the RB gene product, which occurs within 9–24 hr of incubation with activin A; and phosphorylation of RB protein then develops afterward. In addition, these cell cycle‐related events are observed to occur earlier than the accumulation of hemoglobins in K562 cells. It is concluded that transient dephosphorylation of RB protein and prolongation of G1 phase of cell cycle precede and accompany erythroid differentiation caused by activin A and chemical inducers, thus constituting part of the mechanism for induction of differentiation in the erythroleukemia cells. © 1992 Wiley‐Liss, Inc.
Pericellulars substrates of human mast cell tryptase: 72,000 Dalton gelatinase and fibronectin
Authors: Lohi Jouko , Kesksi‐Oja Jorma , Harvima Ilkka
Published: May 2005

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produces a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an Mr 72,000 protein was digested to an Mr62,000 form by human mast cell tryptase while the plasminogen activator inhibitor PAI‐1 was not affected. Cleavage of the Mr72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the Mr 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin‐Sepharose. The soluble form of the Mr72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact Mr72,000 and Mr62,000 degraded form of the protein process gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of Mr72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose‐dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices. © 1992 Wiley‐Liss, Inc.