bluuebunny/crossref_metadata_2025 huggingface dataset:
HMG‐Like chromosomal proteins in Trypanosoma cruzi
Authors: Morales Mónica , Galanti Norbel , Oñate Eddie , Imschenetzky Maria
Published: May 2005

HMG‐like chromosomal proteins from Truypansoma cruzi were studies. Four HMG‐like proteins, designated HMG A, HMGA‐B, HMG‐C, and HMG‐E, were isolated and found to have molecular weights of 35.5 kd, 27.5 kd, 21.8 kd and 10.4 kd, respectively. Immunological relatedness was demonstrated between the mammalian HMG 1,2 and the HMG‐A and HMG‐B from T. cruzi. The relative amount of HMG‐C and HMG‐E proteins vary in T. cruzi depending to the proliferative stage of the cells. HMG‐E protein is increased in proliferating cells when compared to its level in non‐proliferating cells. HMG‐C is increased in the non‐proliferating cells. Probably, the shifts observed in the relative amount of HMG‐like proteins are related to the proliferating cells of this flagellate. The results are consistent with those described for other lower eukaryotes where the HMG‐like proteins isolated are similar but not identical to HMG proteins from vertebrates. © 1992 Wiley‐Liss, Inc.
A human histone H2B.1 Variant gene, located on chromosome 1, utilizes alternative 3′ end processing
Authors: Collart David , Pockwinse Shirwin , Lian Jane B. , Stein Janet L. , Stein Gary S. , Romain Paul L. , Pilapil Suzie , Heubner Kay , Cannizzaro Linda A. , Croce Carlo M.
Published: May 2005

Abstract a variant human H2B histone gene (GL 105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNA regulated differentially during the hela S3 cell cycle. The H2B‐GL105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3′ end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non‐translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B‐GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B‐GL105 histone gene was localized to chromosome region 1q21‐1q23 by chromosomal in situ hybridization and by analysis of rodent‐human somatic cell hybrids using an H2B‐GL105 specific probe. The H2B‐GL105 gene is paired with a functional H2A histone gene and this H2B/H2B gene pair is seperated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF‐1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3′ end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication‐dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation. © 1992 Wiley‐Liss, Inc.
Characteristics of DNA replication in isolated nuclei initiated by an aprotinin‐binding protein
Authors: Coffman Frederick D. , Fresa Kerin L. , Hameed Meera , Cohen Stanley
Published: May 2005

Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin‐binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR‐containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase α (a principal polymerase in DNA replication) but that DNA polymerase β (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease‐treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease‐treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR‐mediated incorporation of 3H‐dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events. © 1993 Wiley‐Liss, Inc.
pH Sensitivity of epidermal growth factor receptor complexes
Authors: Nunez Marissa , Mayo Kevin H. , Starbuck Cindy , Lauffenburger Douglas
Published: May 2005

The association/dissociation binding kinetics of 125I‐labeled mouse epidermal growth factor (EGF) to receptors on human fibroblast cells in monolayer culture have been measured at 4°C as a function of extracellular pH from pH 5–9. At pH 8, steady‐state total binding is maximal. As pH is lowered to 6.5, total binding monotonically decreases dramatically. It changes further only slightly between pH 6.5 and 5 to about 20% of the maximum binding value. Scatchard binding plots at pH 7.5 and above show the commonly observed concave‐upward, non‐linear curve; as pH is lowered, this plot becomes much more linear, indicating that the “high affinity” bound receptor population is greatly diminished. Application of our ternary complex binding model [Mayo et al., J Biol Chem 264:17838–17844, 1989], which hypothesizes complexation of the EGF‐bound receptor with a cell surface interaction molecule, indicates that pH may have some direct effects on ternary complex formation, but the major effect is on EGF‐receptor dissociation. © 1993 Wiley‐Liss, Inc.
Unphosphorylated α‐PKC exhibits phorbol ester binding but lacks protein kinase activity in vitro
Authors: Filipuzzi Ireos , Fabbro Doriano , Imber Roland
Published: May 2005

Expression of the α‐isoform of protein kinase C (α‐PKC) in E. coli yielded the unphosphorylated 74 kD precursor molecule. This precursor form exhibited phospholipid‐ and calcium‐dependent phorbol ester binding but lacked, in contrast to the phosphorylated enzyme, protein kinase activity. In addition, the precursor molecule was found to interact with both threonine and an ATP analogon, which demonstrates that phosphorylation of α‐PKC is not required for binding of substrates, cofactors, or activators. These results, therefore, suggest that posttranslational phosphorylation of α‐PKC is not needed for the formation of a functional enzyme‐substrate complex but is necessary for the catalytic transfer of phosphate residues from ATP to protein substrates.
High resolution distribution of mRNA within the cytoskeleton
Authors: Bassell Gary J.
Published: May 2005

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this research was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems involved. This work required the development of novel in situ hybridization methods for use with electron microscopy.
ABP50: An actin‐binding elongation factor 1α from Dictyostelium discoideum
Authors: Edmonds Brian T.
Published: May 2005

ABP50 is a polypeptide elongation factor 1α from Dictyostelium that is associated with the actin cytoskeleton. Upon chemotactic stimulation, ABP50 undergoes a dramatic cytoplasmic redistribution into newly formed surface projections and in vitro binds to and bundles actin filaments. Many questions are raised by this interaction pertaining to the spatiotemporal regulation of protein synthesis and cytoskeletal organization by extracellular signals.
Effect of cytoarchitecture on the transport and localization of protein synthetic machinery
Authors: Luby‐Phelps Katherine
Published: May 2005

The emerging picture of cytoarchitecture imposes constraints on the transport and localization of several components of the protein synthetic machinery. The range in which “free” polysomes can diffuse through the cytoplasm may be restricted to about 50 nm due to obstruction by cytoskeletal barriers. Individual ribosomes and large transcripts will diffuse at least 4–10 times slower in cytoplasm than in dilute aqueous solution and may be sterically excluded from some cytoplasmic domains. The transport of these components from the nucleus to the cell periphery may be restricted to microtubule‐containing channels that traverse the excluding domains. In the peripheral cytoplasm, mitochondria, endoplasmic reticulum, and other membrane‐bound organelles are found only in nonexcluding channels, while actin, nonmuscle filamin (ABP280), and fodrin are concentrated in excluding domains. This suggests that the cytoplasmic volume may be functionally compartmentalized by local differentiations of cytoarchitecture. Excluding compartments may play a structural role, while nonexcluding compartments are the site of vesicle traffic and protein synthesis.
1α,25‐Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in rat osteosarcoma cells
Authors: Sorensen Ann Marie , Bowman Douglas , Baran Daniel T.
Published: May 2005

1α,25‐Dihydroxyvitamin D3 increases intracellular calcium in rat osteoblast‐like cells that possess the classic receptor (ROS 17/2.8) as well as those that lack the classic receptor (ROS 24/1), indicating that a separate signalling system mediates this rapid nongenomic action. To determine the intracellular sites of this calcium increase, cytosolic and nuclear fluorescence (340 nm/380 nm ratio) were measured in Fura 2AM loaded ROS 17/2.8 cells using digital microscopy. Within 5 min, cytosolic fluorescence increased by 29% (P < 0.05) and nuclear fluorescence by 30% (P < 0.01) after exposure to 1α,25‐dihydroxyvitamin D3 (20 nM). This effect was blocked by the inactive epimer 1β,25‐dihydroxyvitamin D3. In an individual cell, cytosolic and nuclear fluorescence increased gradually after 1, 3, and 5 min exposure to vitamin D. Nuclei were then isolated from ROS 17/2.8 cells to directly measure the hormone's effect on nuclear calcium. The calcium content of Fura 2AM loaded nuclei was not affected by increasing the calcium concentration in the incubation buffer from 50 nM to 200 nM. After 5 min, 1α,25‐dihydroxyvitamin D3, 20 nM, increased the calcium of isolated nuclei in medium containing 50 nM calcium and 200 nM calcium. 1β,25‐dihydroxyvitamin D3, 20 nM, had no effect on nuclear calcium but blocked the 1α,25‐dihydroxyvitamin D3 induced rise in the isolated nuclei. The results indicate that the nuclear membrane of the ROS 17/2.8 cells contain calcium permeability barriers and transport systems that are sensitive to and specific for 1α,25‐dihydroxyvitamin D3.

1α,25‐Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in both intact cells and isolated nuclei suggesting that rapid nongenomic activation of nuclear calcium may play a functional role in osteoblastic activity.
Evidence for calcium mediated conformational changes in calbindin‐D28K (the vitamin D‐induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques
Authors: Leathers Valerie L. , Norman Anthony W.
Published: May 2005

The role of the vitamin D‐induced calcium binding protein termed calbindin‐D (CaBP) in the biological response to 1,25‐dihydroxyvitamin D3 was assessed by photoaffinity labeling techniques. The heterobifunctional cross‐linking reagent methyl‐4‐azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin‐D28K. Calcium‐dependent interactions were evident with purified chick intestinal CaBP‐immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca2+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with MR = 60,000 and 130,000, which were photoaffinity cross‐linked with CaBP in a calcium‐dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol‐specific phospholipase C (PI‐PLC)‐treated supernatant fractions from chick intestinal brush borders. PI‐PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G‐150 chromatography of Triton X‐100 solubilized incubations between calbindin‐D28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin‐D28K undergoes calcium‐dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium‐binding proteins such as calmodulin or troponin C.
Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts
Authors: Collier Nancy C. , Sheetz Michael P. , Schlesinger Milton J.
Published: May 2005

Utilizing video‐enhanced differential interference contrast microscopy of chicken embryo fibroblasts, we observed dramatic changes in the localization and morphology of mitochondria shortly after cells were subjected to a mild heat shock. At normal temperatures mitochondria were distributed in the cell cytoplasm as elongated, tubular, and dynamic organelles but upon heat shock they moved to the perinuclear region and formed a tight ring of short swollen and—in some cases—fused vesicles. Vital dye staining of mitochondria with rhodamine 123 and indirect immunofluorescence staining with antibodies against the mitochondrial‐matrix protein, HSP 60, confirmed these results. Using cells double labeled with antibodies to vimentin and the HSP 60 protein, we found that the changes in mitochondria were accompanied by perturbations of the intermediate filament network that we and others have reported previously for heat shocked cells. Microtubules remained largely unaltered by our heat shock treatment and the redistribution of intermediate filaments and mitochondria occurred even in the presence of taxol, a microtubule stabilizing drug.

The effects of heat shock on mitochondria were reversed when cells were returned to normal temperatures and their recovery to their normal state coincided with return of normal intermediate filament morphology. This recovery was blocked in cells treated with actinomycin D during heat shock, a result indicating that a heat shock protein may be required for recovery. These data are consistent with previously published observations that mitochondria are associated with the intermediate filament network but they extend this interaction to a cell system responding to a physiological stress normally experienced by the intact organism.
Heat‐induced morphological and biochemical changes in the nuclear lamina from Ehrlich ascites tumor cells in vivo
Authors: Krachmarov Chavdar P. , Traub Peter
Published: May 2005

Membrane‐depleted nuclei from Ehrlich ascites tumor (EAT) cells isolated at low ionic strength in the presence of EDTA exhibit highly decondensed chromatin fibers and a loss of morphologically identifiable nucleoli. Treatment of these nuclei with nucleases and 2 M NaCl followed by low‐speed centrifugation permitted the facile isolation of the nuclear lamina layer. Under the same conditions, but after heat‐shock treatment of the living cells, the chromatin appears in a more condensed state, the nucleoli are well‐defined, and the nuclear lamina layer was destabilized in concert with the appearance of an internal nuclear matrix and nucleolar skeleton. Furthermore, we also found both an increase in the protein mass as well as the appearance of a relatively large number of new proteins in this fraction, which are phosphorylated. The major proteins of the nuclear lamina, the lamins, and the residual vimentin remained insoluble. These heat‐shock‐induced changes were also accompanied by a dephosphorylation of lamins A and C but not of lamin B.
Identification of a 170 kDa membrane kinase with increased activity in KB‐V1 multidrug resistant cells
Authors: Sampson Kathleen E. , McCroskey Mark C. , Abraham Irene
Published: May 2005

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of 32P incorporation in proteins that were renatured after being subjected to SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723–20729, 1989; Mol Cell Biol 10:3020–3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB‐V1 cells as compared to the sensitive parental KB‐3‐1 cells. The KB‐V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca++, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P‐glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF‐7/AdrR and murine melanoma B16/AdrR. cells, but the activity was not present in murine leukemia P‐388 sensitive or multidrug resistant cells.
Arrest of cell cycle progression of HeLa cells in the early G1 phase in K+‐depleted conditions and its recovery upon addition of insulin and LDL
Authors: Yamaguchi Hisao , Hosokawa Keiko , Jiang Zheng‐Lin , Takahashi Akira , Ikehara Toshitaka , Miyamoto Hiroshi
Published: May 2005

Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [3H]thymidine. The progression was arrested in a chemically defined medium in which K+ was replaced by Rb+ (Rb‐CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb‐CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K−‐depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase.
Density‐dependent endothelial cell production of an inhibitor of smooth muscle cell growth
Authors: Dodge Andrea B. , Lu Xiaoqing , D'Amore Patricia A.
Published: May 2005

Embryonic data and ultrastructural analyses suggest that the primitive endothelium signals undifferentiated mesenchymal cells to migrate to the forming blood vessel and subsequently regulates mural cell growth and behavior. Upon maturation of the blood vessel, chemotactic and mitogenic signals are apparently diminished and differentiated smooth muscle cells normally remain quiescent. This homeostasis is seemingly upset in conditions which lead to pathologies characterized by smooth muscle cell hyperplasia such as atherosclerosis. By culturing endothelial cells at different densities, we attempted to re‐create the various stages of vascular development. Whereas media conditioned by sparse endothelial cells stimulate smooth muscle cells, media conditioned by dense endothelial cell cultures are inhibitory. Culture of sparse smooth muscle cells in media conditioned for 3 days by postconfluent endothelial cell cultures leads to dose‐dependent and reversible smooth muscle cell inhibition. Furthermore, in the presence of the endothelial cell‐derived inhibitor, smooth muscle cells are rendered refractory to mitogens such as fibroblast growth factor and platelet‐derived growth factor. The inhibitory activity is not attributable to the well‐characterized inhibitors of smooth muscle cell growth, transforming growth factor type‐β, prostaglandin I2, or heparan sulfate proteoglycan. Partial characterization of the inhibitory conditioned media suggests that the active molecule is smaller than 1,000 da, and stable to boiling as well as proteinase K and heparinase digestion. These findings support the concept that there is intercellular communication between endothelial cells and smooth muscle cells and provide evidence for a novel endothelial cell‐derived smooth muscle cell growth inhibitor.
Vimentin serves as a phosphate sink during the apparent activation of protein kinases by okadaic acid in mammalian cells
Authors: Lai Yiu‐Kay , Lee Wen‐Chuan , Chen Kuang‐Den
Published: May 2005

The vimentin contents of four mammalian cell lines originating from rat and human tissues were determined by immunoblotting and scanning densitometry. On per cell volume basis, vimentin content in 9L, KD, and HeLa cells was found to be 206.6, 151.6, and 19.1 ng/μl, respectively. A431 cells were devoid of vimentin. Protein phosphorylation was augmented by treatment of 600 nM okadaic acid for 1 h in these cells. During the apparent activation of protein kinases, vimentin became hyperphosphorylated and the phosphorylation level of other nonvimentin phosphoproteins was relatively little affected in 9L and KD cells. In contrast, cytokeratins and other nonvimentin proteins were heavily phosphorylated in OA‐treated HeLa and A431 cells. Regression analysis indicated that the relative increase in phosphorylation level of nonvimentin phosphoproteins was inversely correlated to the contents of vimentin in the four cell lines [r2 = −0.985]. These observations strongly suggest that vimentin acts as a phosphate sink by which the effects of “excess kinase activity” inflicted by phosphatases inhibition was attenuated.
Vasopressin induces arachidonic acid release through pertussis toxin‐sensitive GTP‐binding protein in aortic smooth muscle cells: Independence from phosphoinositide hydrolysis
Authors: Ito Yoshiaki , Kozawa Osamu , Tokuda Haruhiko , Kotoyori Jun , Oiso Yutaka
Published: May 2005

We previously reported that pertussis toxin (PTX) had little effect on arginine vasopressin‐induced formation of inositol trisphosphate (IP3) in rat aortic smooth muscle cells [Kondo et al.: Biochemical and Biophysical Research Communications 161:677–682, 1989]. In the present study, we investigated the mechanism of vasopressin‐induced arachidonic acid release in rat aortic smooth muscle cells. Vasopressin stimulated both the release of arachidonic acid and the formation of IP3 dose dependently in the range between 10 pM and 1 μM. The effect of vasopressin on arachidonic acid release was more potent than that on the formation of IP3. Quinacrine, a phospholipase A2 inhibitor, significantly suppressed the vasopressin‐induced arachidonic acid release but had little effect on the formation of inositol phosphates. NaF, a GTP‐binding protein activator, mimicked vasopressin by stimulating the arachidonic acid release. The arachidonic acid release stimulated by a combination of vasopressin and NaF was not additive. PTX partially but significantly suppressed the vasopressin‐induced arachidonic acid release. In the cell membranes, PTX catalyzed ADP‐ribosylation of a protein with an Mr of about 40,000. Pretreatment of membranes with 0.1 μM vasopressin in the presence of 2.5 mM MgCl2 and 100 μM GTP markedly attenuated this PTX‐catalyzed ADP‐ribosylation of the protein in a time‐dependent manner. These results strongly suggest that PTX‐sensitive GTP‐binding protein is involved in the coupling of vasopressin receptor to phospholipase A2 in primary cultured rat aortic smooth muscle cells.
Adenovirus genes that modulate the sensitivity of virus‐infected cells to lysis by TNF
Authors: Wold William S. M.
Published: May 2005

TNF is a key inflammatory cytokine with antiviral properties. Human adenoviruses encode several intracellular proteins that mediate the effects of TNF. Expression of the adenovirus immediate early E1A proteins induces viral genes and a host of cellular genes, drives G0 cells into S‐phase, and induces apoptosis and susceptibility to TNF‐induced apoptosis. The adenovirus E1B‐19K protein inhibits both E1A‐ and TNF‐induced apoptosis. The E3‐14.7K protein and the E3‐10.4K/14.5K complex of proteins inhibit TNF‐ but not E1A‐induced apoptosis. The E3 14.7K and 10.4K/14.5K proteins inhibit TNF activation of cytosolic phospholipase A2 (cPLA2), which may explain how they inhibit TNF cytolysis. Since eicosinoids produced from arachidonic acid (the product of cPLA2) are potent mediators of inflammation, the E3 proteins may block the inflammatory response to adenovirus infection. These adenovirus proteins should be novel tools to understand adenovirus pathogenesis, TNF signal transduction, and TNF cytolysis.
Cytokine involvement in viral permissiveness and the progression of HIV disease
Authors: Butera Salvatore T.
Published: May 2005

Many viruses have evolved novel means of exploiting host defense mechanisms for their own survival. This exploitation may be best exemplified by the interrelationships between certain viruses and the host cytokine networks. Many viruses, including the human immunodeficiency virus type‐1 (HIV‐1), rely on the liberation and cellular action of host immune cytokines to expand their host cell range, to regulate their cellular expression, and to maintain their dormant state until the proper extracellular conditions arise. As again exemplified by HIV‐1, viruses may also take an active role regulating cytokine expression and cell surface cytokine receptors. Because the viral life cycle, and in particular the HIV‐1 life cycle, is so intertwined with cytokine regulatory networks, these networks represent potential points for therapeutic intervention. As our understanding of cellular cytokine pathways involved in viral infection and replication continues to expand, so too will our ability to design rational anti‐viral therapies to alter multiple steps along the viral life cycle.
Enhanced repair of a cisplatin‐damaged reporter chloramphenicol‐O‐acetyltransferase gene and altered activities of DNA polymerases α and β, and DNA ligase in cells of a human malignant glioma following in vivo cisplatin therapy
Authors: Ali‐Osman Francis , Berger Mitchel S. , Rairkar Asha , Stein Donna E.
Published: May 2005

Current evidence suggest an important role for increased repair of drug‐induced DNA damage as one of the major mechanisms involved in tumor cell resistance to cis‐DDP. In this study, we examined the DNA repair capacity and the activities of three DNA repair related proteins, namely, DNA polymerases α and β, and total DNA ligase in cells of a malignant oligodendroglioma obtained from a patient before therapy and compared it with those of a specimen of the tumor acquired after the patient had failed cis‐DDP therapy. DNA repair capacity was quantitated as the extent of reactivation of the chloramphenicol‐O‐acetyltransferase (CAT) gene in a eukaryotic expression vector that has been damaged and inactivated by prior treatment with cis‐DDP and then transfected into the tumor cells. The extent of DNA‐platinum adduct formation in the expression vector was determined by flameless atomic absorption spectrometry. The level of cis‐DDP resistance of cells of the two tumors was determined with the capillary tumor stem cell assay. We observed a 2.8‐fold increased capacity to repair Pt‐DNA adducts and reactivate the CAT gene in cells of the tumor obtained after cis‐DDP therapy, compared to cells of the untreated tumor. This was associated with increases of 9.4‐fold and a 2.3‐fold, respectively, in DNA polymerase β and total DNA ligase activities in cells of the treated tumor. At 5 μM cis‐DDP, there was a 5.9‐fold increase in the in vitro cis‐DDP resistance of post‐therapy tumor cells relative to cells of the untreated tumor. No significant difference in DNA polymerase α activity was observed between the two tumors. These data suggest that the enhanced ability to repair cis‐DDP induced DNA damage, mediated, in part, by increased tumor DNA polymerase β and DNA ligase activities, plays an important role in the in vivo acquisition of cis‐DDP resistance in human malignant gliomas, and that these proteins and/or their encoding genes may represent critical targets for strategies to overcome such resistance clinically.
Interferon α selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage
Authors: Briggs R. , Dworkin L. , Briggs J. , Dessypris E. , Stein J. , Stein G. , Lian J.
Published: May 2005

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed constitutively in cells of the myeloid lineage, appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes. The human myeloid leukemia cell lines, HL‐60, U937, and THP‐1, express similar levels of immunochemically detectable MNDA. Although, the level of MNDA mRNA in primary monocytes is very low it was up‐regulated at 6 h following the addition of interferon α. The effect of interferon α on the MNDA mRNA is also observed in the cell lines HL‐60, U937, and THP‐1. The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon α and other agents including interferon γ, endotoxin, poly (I) · poly (C), and FMLP. The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents. Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level. This reduced level of mRNA could then be elevated with subsequent interferon α treatment. The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed. The ability of interferon α to up‐regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes. Granulocyte differentiation induced by retinoic acid treatment of HL‐60 cells did not alter the MNDA mRNA level which was also unchanged following subsequent treatment with interferon α. The lack of interferon α effects on retinoic acid treated HL‐60 cells is consistent with its inability to influence MNDA mRNA level in primary granulocytes.
Identification of transferrin as one of multiple EDTA‐extractable extracellular proteins involved in early chick heart morphogenesis
Authors: Isokawa Keitaro , Rezaee Mehrdad , Wunsch Ann , Markwald Roger R. , Krug Edward L.
Published: May 2005

It was demonstrated previously that a polyclonal antibody (ES1) raised against EDTA extractable proteins from embryonic chicken heart blocks cardiac endothelial‐mesenchymal transformation in a culture bioassay and stains extracellular matrix at sites of embryonic inductive interactions, e.g., developing heart, limb buds, and neural crest forming region (Krug et al., 1987, Dev Biol 120:348–355; Mjaatvedt et al., 1991, Dev Biol 145:219–230). In the present study, by using an antiserum (ES3) to a similar immunogen, we affinity purified four major EDTA‐soluble proteins. These proteins migrated as 27, 44, 63, and 70 kD molecules under reduced conditions and 27, 41, 52, and 59 kD under nonreduced conditions, respectively, on SDS‐PAGE. Based on several criteria, the protein migrating at 70/59 kD (reduced/nonreduced) was indistinguishable from chicken transferrin (conalbumin): (1) amino acid sequencing showed that eight N‐terminal residues were identical to those of chicken transferrin, (2) acid hydrolysates of both proteins had nearly identical compositions, (3) the protein co‐migrated exactly with chicken transferrin under both reduced and nonreduced conditions, and (4) ES3 IgG recognized both the 70/59 kD protein and chicken transferrin by western blot analysis of nonreduced samples, but not with reduced samples. Immunohistochemistry of chicken embryonic heart with antibodies against transferrin demonstrated that anti‐transferrin immunoreactivity is present in myocardium but absent in cardiac endothelium before the initiation of cardiac endothelial‐mesenchymal formation. However, both cardiac endothelium and migrating mesenchymal cells became immunoreactive with anti‐transferrin at the time transformation occurred. These findings suggest a possible involvement of transferrin in the inductive process of cardiac endothelial‐mesenchymal transformation.
Primate testicular histone H1t genes are highly conserved and the human H1t gene is located on chromosome 6
Authors: Koppel Daniel A. , Wolfe Steven A. , Fogelfeld Leon A. , Merchant Peggy S. , Prouty Leonard , Grimes Sidney R.
Published: May 2005

The testis‐specific histone H1t gene is known to be transcribed only in pachytene primary spermatocytes during spermatogenesis. Previous studies of the rat histone H1t gene revealed a unique promoter sequence element between the H1/GC box and the H1/CCAAT box. Proteins in crude nuclear extracts of rat testis bind specifically to this sequence element and a temporal correlation exists between the appearance of these DNA binding proteins and the onset of transcription. These discoveries led to a search for histone H1t genes in other mammalian species. The human and monkey histone H1t genes were amplified from genomic DNA using the polymerase chain reaction (PCR). The amplified genes were cloned and the genomic derived inserts were sequenced using linear PCR. Both proximal promoters contained the highly conserved H1/AC box, H1/CCAAT box, and H1/TATA box found in nongerminal H1 genes. Both promoters also contained the H1/GC box and the H1t/CCTAGG sequence element between the H1/GC box and H1/CCAAT box previously seen only in the H1t promoter. Specific amplification of the human H1t gene using template DNA samples from a NIGMS human/rodent somatic cell hybrid mapping panel has shown that the human histone H1t gene is located on chromosome 6.
WI‐38 cell long‐term quiescence model system: A valuable tool to study molecular events that regulate growth
Authors: Soprano Kenneth J.
Published: May 2005

A number of cell culture model systems have been used to study the regulation of cell cycle progression at the molecular level. In this paper we describe the WI‐38 cell long‐term quiescence model system. By modulating the length of time that WI‐38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G‐1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Through studies aimed at understanding the cause and molecular nature of the prolongation of the prereplicative phase, we have determined that gene expression plays an important role in establishing growth factor “competence” and that once the cell becomes “competent” there is a defined order to the molecular events that follow during the remainder of G‐1. More specifically, we have determined that the prolongation represents a delay in the ability of long term quiescent cells to become fully “competent” to respond to growth factors which regulate progression through G‐1 into S. This prolongation appears to occur as a result of changes during long term quiescence in the ability of immediate early G‐1 specific genes (such as c‐myc) to activate the expression of early G‐1 specific genes (such as ornithine decarboxylase). While ODC is the first and thus far only growth associated gene identified as a target of c‐myc (and the Myc/Max protein complex), it is likely that further studies in this model system will reveal other early G‐1 growth regulatory genes. We anticipate that future follow‐up studies in this model system will provide additional valuable information abuot the function of growth‐regulatory genes in controlling growth factor responsiveness and cell cycle progression.
Requirements for DNA transcription and replication at the beginning of mouse development
Authors: Majumder Sadhan , Depamphilis Melvin L.
Published: May 2005

In mice, the first round of DNA replication occurs in fertilized eggs (1‐cell embryos), while the onset of zygotic gene transcription begins ∼ 20 hours after fertilization, a time that normally coincides with formation of a 2‐cell embryo. One approach to investigating the mechanisms that control these developmentally regulated events has been to microinject plasmid DNA into the nuclei of mouse oocytes and embryos in order to determine the requirements for unique DNA sequences that regulate transcription and replication. The results from these and other studies have revealed two important mechanisms that regulate the beginning of animal development. The first is a time dependent “zygotic clock” of unknown detail that delays the onset of transcription, regardless of whether or not a 2‐cell embryo is formed. The second is a mechanism that represses the activity of promoters and origins of replication specifically in maternal pronuclei of oocytes and 1‐cell embryos, and in all nuclei of 2‐cell embryos, regardless of their parental origin or ploidy. This repression is linked to chromatin, but the striking ability to relieve this repression with specific embryo‐responsive enhancers first appears with formation of a 2‐cell embryo. The need for a TATA‐box to mediate enhancer stimulation of promoter activity appears even later when cell differention becomes evident. Thus, a biological clock delays transcription until both paternal and maternal genomes are replicated and remodeled from a post‐meiotic state to one in which transcription is repressed by chromatin structure in a manner that can be relieved by cell‐specific enhancers at appropriate times during development. © 1994 Wiley‐Liss, Inc.
Transcriptionally active nuclei isolated from intact bone reflect modified levels of gene expression in skeletal development and pathology
Authors: Shalhoub V. , Bortell R. , Jackson M. E. , Marks S. C. , Stein J. L. , Lian J. B. , Stein G. S.
Published: May 2005

Transcriptional regulation of gene expression in vivo in bone, associated with normal development or skeletal disorders, to date, has not been studied. We report the successful isolation of nuclei that are transcriptionally active from normal and osteopetrotic rat bone. Transcription rates of cell growth and bone‐related genes (including histone H4, c‐fos, c‐jun, TGFβ1, β2 macroglobulin, collagen, fibronectin, osteocalcin, osteopontin, and tartrate resistent acid phosphatase) change as a function of calvarial development from birth to 6 weeks and are selectively modified in osteopetrotic animals. Additionally, nuclei isolated from intact bone yield promoter binding factors. Bone nuclei, which transcribe faithfully and contain the normal complement of nuclear protein factors, offer a powerful approach for investigating in vivo gene regulation in skeletal development and pathology. © 1994 Wiley‐Liss, Inc.
Function of osteocytes in bone
Authors: Aarden Elisabeth M. , Nijweide Peter J. , Burger Elisabeth H.
Published: May 2005

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction–coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body–containing lacunae with each other and with the outside world. During differentiation from osteoblast to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are (1) osteocytes are actively involved in bone turnover; (2) the osteocyte network is through its large cell‐matrix contact surface involved in ion exchange; and (3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations. © 1994 Wiley‐Liss, Inc.
Purification and characterization of an activated form of the protein tyrosine kinase Lck from an Escherichia coli expression system
Authors: Flint Nicholas A. , Amrein Kurt E. , Jascur Thomas , Burn Paul
Published: May 2005

The lymphocyte‐specific, nonreceptor protein tyrosine kinase Lck has been purified from an Escherichia coli expression system using a monoclonal antibody column followed by dye‐affinity chromatography. Polyacrylamide gel electrophoretic analysis of purified protein revealed a single 56 kDa band, indicating that recombinant Lck was purified to near‐homogeneity. The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed. Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine‐394), which is characteristic for activated protein tyrosine kinases. Indeed, we found that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy‐terminal tyrosine residue (tyrosine‐505). Thus, we have overproduced recombinant human Lck in E. coli and developed a simple two‐step purification procedure which yields highly active enzyme. This will enable the identification and characterization of potential regulators and targets of Lck and thereby greatly facilitate studies which will clarify its role in T cell signal transduction. © 1994 Wiley‐Liss, Inc.
Comparative effects of hepatocyte growth factor and epidermal growth factor on motility, morphology, mitogenesis, and signal transduction of primary rat hepatocytes
Authors: Stolz Donna Beer , Michalopoulos George K.
Published: May 2005

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are major hepatacyte mitogens, but HGF, also known as scatter factor (SF), has also been shown as a potent motogen for epithelial and endothelial cells. The mechanisms by which HGF is a stronger motogen compared to other mitogens are not understood. Here we report a comparative study of the effect of the two growth factors on cultured primary rat hepatocytes regarding their differential effects on morphology, mitogenicity, and motility as well as the phosphorylation of cytoskeletal‐associated proteins. Using three different motility assays, both HGF and EGF increased the motility of hepatocytes, but HGF consistently elicited a significantly greater motility response than EGF. Additionally, HGF induced a more flattened, highly spread morphology compared to EGF. To examine if HGF and EGF phosphorylated different cytoskeletal elements as signal transduction targets in view of the observed variation in morphology and motility, primary cultures of 32P‐loaded rat hepatocytes were stimulated by either HGF or EGF for up to 60 min. Both mitogens rapidly stimulated four isoforms of MAP kinase with similar kinetics and also rapidly facilitated the phosphorylation of cytoskeletal‐associated F‐actin. Two cytoskeletal‐associated proteins, however, were observed to undergo rapid phosphorylation by HGF and not EGF during the time points described. One protein of 28 kDa was observed to become phosphorylated fivefold over controls, while the EGF‐stimulated cells showed only a slight increase in the phosphorylation of this protein. Another protein with an apparent mwt of 42 kDa was phosphorylated 20‐fold at 1 min and remained phosphorylated over 50‐fold over control up to the 60 min time point. This protein was observed to become phosphorylated by EGF only after 10 min, and to a lesser extent (20‐fold). Taken together, the data suggest that HGF and EGF stimulate divergent as well as redundant signal transduction pathways in the hepatocyte cytoskeleton, and this may result in unique HGF‐ or EGF‐specific motility, morphology, and mitogenicity in hepatocytes. © 1994 Wiley‐Liss, Inc.
Induction of c‐fos and c‐jun mRNA at the M/G1 border is required for cell cycle progression
Authors: Cosenza Stephen C. , Yumet Gladys , Soprano Dianne Robert , Soprano Kenneth J.
Published: May 2005

The proto‐oncogenes c‐fos and c‐jun have been shown in numerous model systems to be induced within minutes of growth factor stimulation, during the G0/G1 transition. In this report we use the mitotic shake‐off procedure to generate a population of highly synchronized Swiss 3T3 cells. We show that both of these immediate‐early, competence genes are also induced during the M/G1 transition, immediately after completion of mitosis. While c‐fos mRNA levels drop to undetectable levels within 2 hr after division, c‐jun mRNA levels are maintained at a basal level which is ∼ 30% maximum throughout the remainder of G1. In order to access the functional significance of these patterns of c‐fos and c‐jun expression, antisense oligodeoxynucleotides specific to c‐fos or c‐jun were added to either actively growing Swiss 3T3 cells or mitotically synchronized cells, and their ability to inhibit DNA synthesis and cell division determined. Our results show that treatment of Swiss 3T3 cells with either c‐fos or c‐jun antisense oligodeoxynucleotides, while actively growing, during mitosis, or in early G1, results in a reduction in ability to enter S and subsequently divide. This was also true if Swiss 3T3 cells were treated during mid‐G1 with c‐jun antisense oligodeoxynucleotides. These results demonstrate that the regulation of G1 progression following mitosis is dependent upon the expression and function of the immediate‐early, competence proto‐oncogenes c‐fos and c‐jun. © 1994 Wiley‐Liss, Inc.
Overcoming obstacles to metastasis ‐ defenses against host defenses: Osteopontin (OPN) as a shield against attack by cytotoxic host cells
Authors: Denhardt David T. , Chambers Ann F.
Published: May 2005

Osteopontin (OPN) serves both a cell attachment function and a cell signalling function via the αvβ3 integrin, in its cell attachment capacity it can promote attachment of both osteolasts to bone hydroxyapatite and various other cell types to basement membrane/extracellular matrix. In its cell signalling capacity it initiates a signal transduction cascade that includes changes in the intracellular calcium ion levels and the tyrosine phosophorylation status of several proteins including paxillin. Effects on gene expression include suppression of the induction of nitric oxide synthase by inflammatory mediators. OPN can also reduce cell oxidant and inhibit the killing of tumor cells by activated macrophages and endothelial cells. We hepothesize that those cancer cells that produce OPN at elevated levels can suppress the oxidative burst, inhibit NO production, and thus protect themselves from killing by specific host cell types.