bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three‐dimensional growth and differentiation of ovarian tumor cell line in high aspect rotating‐wall vessel: Morphologic and embryologic considerations
Authors: Becker Jeanne L. , Prewett Tacey L. , Spaulding Glenn F. , Goodwin Thomas J. 
Published: May 2005

AbstractCancer of the ovary is the leading cause of death from gynecologic malignancy. To understand better these aggressive tumors, the development of in vitro models to study human ovarian cancer is critical. However, the establishment of long‐term cell lines has been difficult, due to the generalized poor survival of patient tumor cells grown in primary culture. Satisfactory culture systems for ovarian tumor cells have therefore been limited. To study cellular interactions involved in the growth and differentiation of these tumors, a cell line was established from a mixed müllerian tumor of the ovary. This cell line, designated LN1, was cultured on microcarrier beads in the high aspect rotating‐wall vessel. The tumor cells grown in this vessel readily proliferated without a requirement for cocultivation with a supportive cell layer. Evaluation of cellular morphology by phase contrast light microscopy and scanning electron microscopy revealed the presence of three‐dimensional multicellular aggregates consisting of multiple cell‐coated beads bridged together, as well as scattered aggregates of LN1 cells proliferating as spheroids free in suspension. In contrast to conventional culture systems, culture in the high aspect rotating‐wall vessel facilitated the generation of multiple cell types that could be recovered. These results illustrate the ability of this culture system to provide the biological conditions necessary for pluripotent cell growth. © 1993 Wiley‐Liss, Inc.
Prospects for use of microgravity‐based bioreactors to study three‐dimensional host—tumor interactions in human neoplasia
Authors: Jessup J. M. , Goodwin T. J. , Spaulding G. 
Published: May 2005

AbstractMicrogravity offers unique advantages for the cultivation of mammalian tissues because the lack of gravity‐induced sedimentation supports three‐dimensional growth in batch culture in aqueous medium. Bioreactors that simulate microgravity but operate in unit gravity provide conditions that permit human epithelial cells to grow to densities approaching 107 cells/ml on microcarriers in suspension, in masses up to 1 cm in diameter, and under conditions of low shear stress. While useful for many different applications in tissue culture, this culture system is especially useful for the analysis of the microenvironment in which host matrix and cells interact with infiltrating tumor cells. Growth in the microgravity‐based bioreactor has supported morphological differentiation of human colon carcinoma cells when cultured with normal human stromal cells. Furthermore, these co‐cultures produced factors that stimulated goblet cell production in normal colon cells in an in vivo bioassay. Early experiments also suggest that the microgravity environment will not alter the ability of epithelial cells to recognize and associate with each other and with constituents of basement membrane and extracellular matrix. These findings suggest that cells grown in bioreactors that simulate aspects of microgravity or under actual microgravity conditions will produce tissues and substances in sufficient quantity and at high enough concentration to promote characterization of molecules that control differentiation and neoplastic transformation. © 1993 Wiley‐Liss, Inc.
A point mutation in the 5′ splice region of intron 7 causes a deletion of exon 7 in adenosine deaminase mRNA
Authors: Kawamoto Hiroshi , Ito Kazuhiko , Kashii Saburo , Monden Sumie , Fujita Masahiro , Norioka Mihoko , Sasai Yoshiki , Okuma Minoru 
Published: May 2005

AbstractAn adenosine deaminase (ADA;EC 3.5.4.4)‐deficient B lymphoblastoid cell line BAD05 derived from a Japanese patient with severe combined immunodeficiency was characterized. As previously reported, one allele of BAD05 expresses undetectable ADA mRNA, and the other allele produces an aberrant mRNA without exon 7. Genomic ADA DNA of BAD05 spanning from a portion of exon 6 to a portion of exon 8 was amplified by PCR. The amplified fragments were cloned into a vector, and 8 clones were isolated and sequenced. The analytical result showed a single base change of G to A at the invariant 5′ GT of intron 7 of ADA gene in one allele of BAD05, which accounts for the elimination of exon 7 during splicing. © 1993 Wiley‐Liss, Inc.
Binding and subcellular distribution of cyclosporine in human fibroblasts
Authors: Tipton David A. , Dabbous Mustafa Kh. 
Published: May 2005

AbstractThe uptake, binding, and subcellular sites of accumulation of [3H]‐cyclosporine (CS) in two human gingival fibroblast strains, GN 23 and GN 54, have been examined. GN 23 responds to CS treatment with a decrease in collagenolysis, while GN 54 does not. Binding of the drug was determined using [3H]‐CS concentrations ranging from 10−5 to 10−8 M in the absence or presence of excess unlabeled CS (1 mM). The binding of the drug to both strains was specific and reached a plateau within 10 min, remaining at that level for up to 1 h. Scatchard analysis of the specific binding of [3H]‐CS to the responsive GN 23 strain revealed two dissociation constants: KD = 5 × 10−8 M (1.2 × 107 sites/cell) and KD = 1.4 × 10−6 M (2.2 × 108 sites/cell). GN 54, on the other hand, had only one class of low affinity binding site (KD = 0.47 × 10−6 M [1.2 × 108 sites/cell]). Unlabeled CS (0.01–1 mM) inhibited the binding of [3H]‐CS in a dose‐dependent manner to both strains, as did the calmodulin antagonist W‐7, to a lesser extent. However, W‐7 inhibited CS binding much more efficiently in GN 54 than in GN 23, suggesting that calmodulin may be the predominant CS receptor in GN 54. In both strains, 70% of the drug accumulated in the crude nuclear fraction after a 1 min incubation, with very little (⩽ 4%) being membrane associated, and the remainder was in the cytosol. In GN 23, CS levels in the crude nuclear fraction reached 80% by 20 min, and remained at this level for up to 1 h. In contrast, in GN 54, at incubation times of more than 1 min, the drug did not selectively accumulate in the crude nuclear fraction, but appeared to be in equilibrium between the nuclear and cytosolic fractions. These data show that the CS resistance of human gingival fibroblasts was not due to their inability to take up and bind CS. Rather, the different effects of CS on the collagenolysis of the responder and non‐responder fibroblast strains may be related to the types of CS receptors they possess and differences in the cellular metabolism of CS occurring after binding, including the subcellular sites of drug accumulation. © 1993 Wiley‐Liss, Inc.
Homeodomain protein binding sites, inverted repeats, and nuclear matrix attachment regions along the human β‐globin gene complex
Authors: Boulikas Teni 
Published: May 2005

Abstractβ‐Globin genes in primates arose during evolution by duplication of an ancestral gene, and their order of arrangement along the DNA is related to their timing of expression during development. We believe that nuclear matrix anchorage sites (MARs) along the β‐globin gene complex considered to be mass binding sites for transcription protein factors, some of which are developmental stage specific and others ubiquitous, play a decisive role in cell memory by determining the developmental stage‐specific expression of the genes. The AT‐rich class of MARs appears to possess a significant number of ATTA and ATTTA motifs known to be mass binding sites for homeodomain proteins that determine body formation in development. MARs also appear to harbor origins of replication, to be enriched in inverted repeats (dyad symmetry motifs) and were proposed to include the DNase I hypersensitive sites of a particular gene determined at the chromatin level. This study is an attempt to finely identify MARs at the nucleotide level along the β‐globin gene complex. Searches of a contiguous stretch of about 73.3 kb of human sequences comprising and surrounding the ε‐, γG‐, γA‐, δ‐, and β‐globin genes of the human β‐globin gene complex for homeotic protein binding sites as well as for inverted repeats has shown that these elements are clustered nonrandomly at particular sites within the β‐globin gene complex. These sites are presumed to be the AT‐rich class of MARs of the β‐globin gene complex. The inverted repeats which are characteristic of origins of replication and some promoter/enhancer regions and the homeotic protein sites seem to include the DNase I hypersensitive sites of the gene complex. Indeed, dyad symmetry sequences are present close to the four DNase I HS sites in the locus control region (LCR) of the gene complex as well as in the 5′ flanking regions and the large introns of the δ‐ and β‐globin genes. A search of the putative MAR regions of the gene complex suggests that, in addition to their enrichment in ATTA motifs, palindromes, and DNase I hypersensitive sites, these regions may comprise TG‐rich motifs and potential Z‐DNA as well as polypurine and polypyrimidine blocks.From the positions of palindromes and clusters of homeodomain protein sites along the complex we propose that an extended origin of replication able to initiate at several sites is present in the LCR and two others surrounding the δ‐ and β‐globin genes. Furthermore, we propose that DNase I HS sites, potential Z‐DNA, polypurine and polypyrimide stretches, TG‐boxes, homeodomain protein sites, and dyad symmetry motifs may be features diagnostic of MARs. This analysis supports a model which predicts that facultative matrix anchorage sites containing homeotic and other transcription protein factor binding sites might anchor the ε‐, γ‐, and β‐globin genes in embryonic, fetal, and adult tissue, respectively, and might thus regulate the ordered developmental expression of the genes in the β‐globin gene complex.
Signal transduction mechanisms of recombinant bovine neurokinin‐2 receptor stably expressed in Baby hamster kidney cells
Authors: Eistetter Harald R. , Mills Ann , Arkinstall Stephen J. 
Published: May 2005

AbstractThe bovine neurokinin‐2 (NK‐2) receptor gene was stably transfected into Baby hamster kidney (BHK‐21) fibroblasts and one recombinant clone expressing 17,700 high‐affinity [125I]neurokinin A (NKA) binding sites/cell characterized further. [125I]NKA binding was displaced by unlabeled NKA with an IC50 of 8.26 ± 2 nM (n = 5) and with the rank order of potency NKA > neurokinin B (NKB) > Substance P (SP) confirming pharmacological characteristics of an NK‐2 receptor subtype. Stimulation with NKA resulted in a rapid and dose‐dependent increase in inositol 1,4,5‐trisphosphate (IP3) levels (EC50 = 32 ± 10 nM; n = 7) which was paralleled by a transient biphasic rise in intracellular free calcium concentration [Ca2+]i (EC50 = 35 ± 20 nM; n = 3). In addition to phosphoinositide (PI) hydrolysis and Ca2+ mobilization, NKA was found to stimulate both cyclicAMP formation (EC50 = 1.02 ± 0.26 μM; n = 7) and [3H]arachidonic acid mobilization (EC50 = 0.65 ± 0.45 μM; n = 4). Interestingly, cyclicAMP levels also rose after addition of an exogenous arachidonic acid metabolite, prostaglandin E2 (PGE2) (EC50 = 11.5 ± 2 μM). Similar observations of NKA‐induced IP3 production, Ca2+ mobilization, arachidonic acid liberation, and cAMP formation have been made previously following expression of the bovine NK‐2 receptor in Chinese hamster ovary (CHO) epithelial cells. The present results suggest that activation of NK‐2 receptors leads to characteristic and reproducible intracellular second messenger responses in a subclass of cell types which includes fibroblasts and epithelial cells irrespective of their genetic and phenotypic background.
Expression of type IV collagen‐degrading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis
Authors: Karakiulakis G. , Papakonstantinou E. , Maragoudakis M. E. , Misevic G. N. 
Published: May 2005

AbstractType IV collagen‐degrading activity was expressed in homogenates of Lytechinus pictus embryos during embryogenesis. Activity was concentrated 1,600‐fold by ammonium sulfate fractionation, ion exchange, and gel chromatography and could not be activated further upon trypsin or organomercurial treatment. This enzyme activity could also degrade gelatin but had no affinity for type I, III, and V collagens. Activity was inhibited by addition of excess type IV collagen or gelatin, but was unaffected by addition of excess amounts of non‐collagenous proteins of the extracellular matrix. Chelators such as 1,10‐phenanthroline or Na2EDTA reduced activity to control levels. Inhibitors of plasmin and of serine and thiol proteases were without effect. Type IV collagen‐degrading activity first became apparent at the stage of early mesenchyme blastula. It then increased by a small increment and remained stable up to the stage of late mesenchyme blastula, coinciding with first detection of collagen synthesis and the appearance of the archenteron. Thereafter, a sharp increase in activity was observed, concurrently with remodelling of the archenteron. Maximum activity was attained at prism stage and was retained throughout to pluteus‐larva stage. The specific inhibitors of collagen biosynthesis 8,9‐dihydroxy‐7‐methyl‐benzo[b]quinolizinium bromide and tricyclodecane‐9‐yl xanthate arrested sea urchin embryo development at early blastula, prevented the invagination of the archenteron, and reverted the expression of type IV collagen‐degrading activity to non‐detectable levels. Removal of the inhibitors allowed embryos to gastrulate and express type IV collagen‐degrading activity.
ADP‐ribosylation of nucleolar proteins in HeLa tumor cells
Authors: Leitinger Norbert , Wesierska‐Gadek Jozefa 
Published: May 2005

AbstractADP‐ribosylation reactions in nucleoli of exponentially growing HeLa cells were studied. Isolated nuclei or nucleoli were labeled with 32P‐NAD; then the nucleolar proteins were analyzed by 1‐dimensional and 2‐dimensional polyacrylamide gel electrophoresis (PAGE) and modified proteins were detected by autoradiography. The labeled nucleolar proteins were also chromatographically fractionated on DEAE‐cellulose. Electrophoretic analysis of total nucleolar and chromatographically purified proteins revealed that besides nuclear ADP‐ribosyltransferase and histones two characteristic nucleolar phosphoproteins numatrin/B23 and nucleolin/C23 were modified by ADP‐ribosylation.
1,25‐Dihydroxyvitamin D3 increases type 1 interleukin‐1 receptor expression in a murine T cell line
Authors: Lacey D. L. , Erdmann J. M. , Tan H.‐L. 
Published: May 2005

AbstractThe biologically active metabolite of vitamin D3, 1,25 (OH)2 D3, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and 125I‐interleukin 1α, we show that 1,25 (OH)2 D3 (5,50 nM) enhanced 125I‐interleukin 1α binding up to almost 2‐fold over control. This 1,25 (OH)2 D3 effect occurred in a dose‐dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of 125I‐interleukin 1α binding data demonstrated that 1,25 (OH)2 D3 enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D3, 25 (OH) D3, also augmented 125I‐interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)2 D3. This observation, combined with the presence of high‐affinity 3H‐1,25 (OH)2 D3 receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti‐type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)2 D3‐enhanced 125I‐interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)2 D3 enhanced interleukin 1 receptor expression. Northern blots hybridized with a 32P‐type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)2 D3 enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)2 D3 pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)2 D3's role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.
Induction of bone‐related proteins, osteocalcin and osteopontin, and their matrix ultrastructural localization with development of chondrocyte hypertrophy in vitro
Authors: Lian J. B. , McKee M. D. , Todd A. M. , Gerstenfeld Louis C. 
Published: May 2005

AbstractEndochondral bone formation occurs by a series of developmentally regulated cellular events from initial formation of cartilage tissue to stages of calcified cartilage, resorption, and replacement by bone tissue. Several studies have raised the question of the possibility that the hypertrophic chondrocytes associated with the calcifying cartilage matrix can acquire properties similar to osteoblasts. We have addressed this possibility by measuring synthesis within hypertrophic chondrocytes in vitro of two bone‐related proteins, osteopontin and osteocalcin. Chondrocytes derived from chick embryo ventral vertebral tissue were cultured under conditions that promoted extracellular matrix mineralization and differentiation towards the hypertrophic phenotype as indicated by the induction of Type X collagen, alkaline phosphatase, and diminished expression of Type II collagen and the core protein of large proteoglycan. In these cultures, osteopontin synthesis was detected in early cultures in the absence of a calcified matrix; in contrast, an absence of the bone‐specific protein osteocalcin was observed. However, with onset of development of the hypertrophic phenotype an induction of protein expression for osteocalcin was observed with a significant (twofold) increase in osteopontin. Maximal levels of osteocalcin synthesis occurred with the peak of alkaline phosphatase activity and Type X collagen mRNA levels. The levels of osteocalcin synthesis were induced fiftyfold from the earliest level of detection but this level was only one one‐hundredth of that observed for mature chick osteoblast cultures. Osteocalcin and osteopontin were characterized by several criteria (electrophoresis, immunoblotting, chromatographic characteristics, and response to 1,25(OH)2D3) which confirmed their molecular properties as being identical to osteoblast synthesized proteins. The coordinate change in the cellular phenotype to the hypertrophic chondrocyte was shown to be concurrent with ultrastructural maturation of the cells and the accumulation of osteocalcin and osteopontin in the extracellular matrix associated with hydroxyapatite at sites of mineralization. Since the ultrastructural features of the cells in vitro and the extracellular matrix surrounding the lacunae have features of the hypertrophic chondrocyte and associated matrix in vivo, the induction of the bone‐specific protein osteocalcin suggests that at least a population of these cells may develop osteoblastic phenotypic markers in association with mineralizing matrix. The detection of osteocalcin and the high level of synthesis of osteopontin may represent an advanced stage of chondrocyte hypertrophy or the possibility of a trans‐differentiation of the chondrocytes to an osteoblastic‐like cell.
Effect of gallium nitrate in vitro and in normal rats
Authors: Jenis L. G. , Waud C. E. , Stein G. S. , Lian J. B. , Baran D. T. 
Published: May 2005

AbstractGallium nitrate (GN) is an inhibitor of bone resorption and thereby may result in a change in coupled bone formation. In the present investigation the effects of GN on bone formation were studied in the rat osteosarcoma (ROS) 17/2.8 cell line and normal diploid rat osteoblasts (ROB) in vitro and the femur of rats treated in vivo, measuring mRNA levels for two osteoblast parameters, type I collagen, a marker of matrix formation, and osteocalcin, a bone specific protein and also histone H4, a marker of cell proliferation. GN, at 50 μM for 3 h, increased type I collagen mRNA levels by 132% in ROS 17/2.8 cells and by 122% in proliferating ROB cells. Osteocalcin (OC) mRNA levels were decreased by 61% in ROS 17/2.8 cells and by 97% in differentiated ROB cells. These changes occurred in the absence of any effects on cell proliferation. Seventy‐day‐old female rats were then treated with GN, 0.5 mg/kg/day, for 3 weeks. As previously reported, GN decreased serum calcium levels, but had no effect on lumbar or femoral bone density. In contrast to the in vitro effects, GN had no effect on type I collagen steady‐state mRNA levels in the femur; however, it decreased OC steady‐state mRNA levels in the femur by 58%. These results suggest that GN has similar in vitro effects in transformed and normal osteoblasts, while the collagen‐stimulatory effects observed in vitro cannot be extrapolated to in vivo models. The consistent inhibition of osteocalcin in vitro and in vivo suggests a more specific target for GN that may relate to its effects in inhibiting bone resorption in normal rats.
Platelet‐derived growth factor induces proliferation of hyperplastic human prostatic stromal cells
Authors: Vlahos Chris J. , Kriauciunas Tracey D. , Gleason Philip E. , Jones Jeffrey A. , Eble John N. , Salvas Dan , Falcone Julie F. , Hirsch Kenneth S. 
Published: May 2005

AbstractProstatic hyperplasia (BPH) is a very common disease in elderly men and is characterized by abnormal proliferation of the stromal and epithelial cells of the prostate. The observation that BPH often occurs in association with chronic inflammation has led to the examination of the possibility that platelet‐derived growth factor (PDGF), which is released in response to inflammation, may be an etiological factor in the genesis of the disease. It has been shown that cultured cells derived from human prostatic tissue express high affinity PDGF‐β receptors based on receptor binding and cross‐linking studies with [125I]‐PDGF‐BB. The experiments presented below demonstrate that PDGF receptors are activated in response to the growth factor and that mitogenesis is induced. PDGF‐BB treatment of cultured human prostate cells derived from patients with BPH activates the signal transduction pathway of the PDGF receptor as shown by the presence of several phosphoproteins in antiphosphotyrosine immunoprecipitates, including autophosphorylation of the PDGF receptor. Phosphatidylinositol (PI) 3‐kinase activity is also increased in cells stimulated with PDGF. The addition of PDGF‐BB to the medium causes a variable but dose‐dependent increase in [3H]‐thymidine incorporation. This paper describes the first demonstration that PDGF is a potent mitogen for human cells derived from patients exhibiting prostatic hyperplasia, and also demonstrates that the cellular response to PDGF‐BB is heterogeneous in a manner that is consistent with the varying degree of hyperplasia and inflammation clinically and histologically in the tissue specimens.