bluuebunny/crossref_metadata_2025_split huggingface dataset:
Long‐term marrow cultures: human and murine systems
Authors: Quesenberry Peter , Temeles Daniel , McGrath Helen , Lowry Philip , Meyer David , Kittler Ellen , Deacon Donna , Kister Kay , Crittenden Rowena , Srikumar Kotteazeth 
Published: May 2005

AbstractThe intramedullary control of marrow cell production has been a difficult area to approach experimentally. The introduction by Dr. Dexter and colleagues of long‐term stromal dependent culture systems for murine marrow and the adaptation of these systems to human marrow growth have allowed for in‐vitro studies of stromal dependent hemopoiesis. Despite some controversy in this area, most studies appear to show that adherent murine or human stromal cells are capable of producing a relatively large number of hemopoietic growth factors including G‐CSF, GM‐CSF, CSF‐1, IL‐6 and, at least by PCR analysis, IL‐3. Other work indicates that the most primitive hemopoietic cells which appear to be multifactor responsive adhere directly to these stromal cells presumably through mediation of various adherence proteins.An early acting, multilineage factor termed hemolymphopoietic growth factor‐1 (HLGF‐1) has been isolated from a murine stromal cell line and may be identical to the recently described ligand for the c‐kit receptor. This may represent an important early survival/maintenance factor for stem cells in this system.Studies on primitive stem cells, especially the high proliferative potential colony forming cell (HPP‐CFC), indicate that they are responsive to varying combinations of growth factors and that with increasing numbers of growth factors, as studied in serum‐free systems, decreasing concentrations of the factors may be biologically active.These observations altogether suggest that intramedullary hemopoiesis may be regulated by the positioning of early multifactor responsive stem cells via adherent proteins in juxtaposition to synergistically acting combinations of grwoth factors attached to stromal cell surfaces or the extracellular matrix. In addition, selective production of different growth factors from different subsets of cells may create growth factor gradients and explain the spacial distribution of different cell types within the marrow cavity.
A role for osteocalcin in osteoclast differentiation
Authors: Glowacki J. , Rey C. , Glimcher M. J. , Cox K. A. , Lian J. 
Published: May 2005

AbstractSpecific cellular interactions with components of the extracellular matrix can influence cellular differentiation and development of many tissues. The extracellular matrix of bone is composed of organic constituents and a solid phase of calcium and inorganic phosphate (apatite). When implanted subcutaneously in rats, particles of bone matrix (BPs) recruit progenitors that differentiate into multinucleated cells with osteoclastic features. Because BPs deficient in osteocalcin, a bone matrix protein, were less efficient at promoting osteoclast formation than were normal BPs, we directly examined the influence of osteocalcin on osteoclast differentiation. We evaluated tissue responses to particles of synthetic crystalline apatite alone (Ap), having many of the features of native apatite of mature bone, or to apatite prepared with osteocalcin (Ap/OC), bovine serum albumin (Ap/BSA) or rat bone collagen (Ap/Col). Twelve days after subcutaneous implantation in normal rats, Ap, Ap/BSA, and Ap/Col particles generated a mild foreign body reaction with multinucleated cells in direct contact with the particles; these cells were negative for tartrate‐resistant acid phosphatase (TRAP) activity and lacked ruffled borders. In contrast, Ap particles containing approximately 0.1% osteocalcin were partially resorbed and they generated more multinucleated cells that were TRAP‐positive, were immunoreactive with an antibody against tartrate‐resistant purple acid phosphatase, and displayed ultrastructural features of active osteoclasts including ruffled borders and clear zones. These data support the hypothesis that osteocalcin may function as a matrix signal in the recruitment and differentiation of bone‐resorbing cells.
Pristane induced effects on chromatin of rat lymphoid cells
Authors: Garrett Lenora R. , Cuchens Marvin A. 
Published: May 2005

AbstractThe effects of pristane on the conformation of chromatin in cells isolated from the lymphoid tissues of pristane‐treated Copenhagen rats were examined by flow cytometry, thermal denaturation, sensitivity to enzymatic digestion, and histone protein analyses. Decreases were observed in the fluorescent intensities of propidium iodide (PI) stained nuclei isolated from lymphoid cells of pristane‐treated rats when compared with normal rat lymphoid nuclei. Studies to address the possible basis for the pristane‐induced changes in the DNA staining characteristics of lymphocytes demonstrated that (1) there were no decreases in the amount of DNA present in the nuclei, (2) nuclei isolated from pristane treated rats were less sensitive to thermal denaturation, as well as DNase I enzymatic digestion, and (3) there were apparent increases in the expression of the H1 histone proteins. Collectively, these results suggest that pristane elicits a conformational change in the chromatin which may be mediated by altered expression of nuclear‐associated histone proteins.
The IL‐2 mediated amplification of cellular cytotoxicity
Authors: Grimm Elizabeth Ann , Owen‐Schaub Laurie 
Published: May 2005

AbstractHigh dose [> 1 nM or 30 IU] interleukin‐2 (IL‐2) can induce MHC ‐unrestricted killing from various lymphoid populations. Although it is well established that CD16+ NK cells are the major source of blood‐derived LAK precursors, other lymphoid cells, including several CD3+ T subsets can be a source of precursor activity. We hypothesize that most, if not all, lymphocytes with cytolytic potential may eventually express MHC‐unrestricted killing, when provided with adequate IL‐2 to intiate required secondary cytokine production. This perspective article presents our cumulative data supporting the role of secondary cytokines in the IL‐2 initiated activation of MHC‐unrestricted killing, first by our observations of synergy with the exogenously added TNFs or IL‐1s in combination with low dose IL‐2, and then by the evidence of endogenous cytokine production and response in lymphocytes stimulated with high dose IL‐2.Understanding the amplification mechanism(s) of the various effector arms of the immune system is critical to the eventual regulation of graft rejection, autoimmune phenomena, and potentially to the treatment of cancer. Our studies have focused on the cytotoxic lymphocyte effector system, and have addressed the molecular pathways by which IL‐2 induced cytokines influence the quantity and quality of the cytotoxic lymphocyte response.This article will review the pivotal role that IL‐2 plays in the development of CTL (MHC‐restricted antigen‐specific cytotoxic lymphocytes), followed by the description of how studies in the CTL system led to the observation that IL‐2 alone can activate a heterogeneous collection of MHC‐unrestricted killer lymphocytes, originally known as “Lymphokine Activated Killers” or LAK. We will then describe experiments performed in out laboratory over the past several years demonstrating the positive regulation of LAK activity by exogenous addition of TNF‐α, TNF‐β, IL‐1α or IL‐1β. Finally, we will summarize our data and propose, in the context of the current literature, the endogenous autocrine/paracrine amplification network of secondary cytokines operative in the generation of LAK.
Long‐term expression of gene introduction into normal human T‐lymphocytes by retroviral‐mediated gene transfer
Authors: Fauser A. A. 
Published: May 2005

AbstractHuman T‐lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T‐cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T‐lymphocytes were propagated in culture for up to 14 weeks with interleukin‐2 (IL‐2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T‐cells are capable of long‐term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.
Characterization of Synthetic Peptide Substrates for p34cdc2 Protein Kinase
Authors: Marshak Daniel R. , Vandenberg Mark T. , Bae Young Seuk , Yu Ii Je 
Published: May 2005

AbstractSynthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H‐A‐D‐A‐Q‐H‐A‐T‐P‐P‐K‐K‐K‐R‐K‐V‐E‐D‐P‐K‐D‐F‐OH (T antigen) and H‐K‐R‐A‐L‐P‐N‐N‐T‐S‐S‐S‐P‐Q‐P‐K‐K‐K‐P‐L‐D‐G‐E‐Y‐NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 μM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexs of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2‐binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinases and their regulation throughout the cell division cycle.
A combination of posttranslational modifications is responsible for the production of neuronal α‐tubulin heterogeneity
Authors: Eddé Bernard , Rossier Jean , Caer Jean‐Pierre Le , Berwald‐Netter Yoheved , Koulakoff Annette , Gros François , Denoulet Philippe 
Published: May 2005

AbstractWe describe the presence of α‐tubulin and MAP2 acetyltransferase activities in mouse brain. The enzyme(s) copurified with microtubules through two cycles of assembly‐disassembly. Incubation of microtubule proteins with [3H]acetyl CoA resulted in a strong labeling of both α‐tubulin and MAP2. To determine the site of the modification, tubulin was purified and digested with Glu‐C endoproteinase. A unique radioactive peptide was detected and purified by HPLC. Edman degradation sequencing showed that this peptide contained €N‐acetyllysine at position 40 of the α‐tubulin molecule. This result demonstrates that mouse brain α‐tubulin was acetylated at the same site as in Chlamydomonas. Isoelectric focusing analysis showed that acetylated α‐tublin was resolved into five isoelectric variants, denoted α3 and α5 to α8. This heterogeneity is not due to acetylation of other sites but results from a single acetylation of Lys40 of an heterogeneous population of α‐tubulin isoforms. These isoforms are produced by posttranslational addition of one to five glutamyl units. Thus, neuronal α‐tubulin is extensively modified by a combination of modifications including acetylation, glutamylation, tyrosylation, and other yet unknown modifications.
Fibroblast heterogeneity in collagenolytic response to cyclosporine
Authors: Tipton David A. , Stricklin George P. , Dabbous Mustafa Kh. 
Published: May 2005

AbstractTo investigate the mechanism of cyclosporine (CS)‐induced fibrotic gingival enlargement, the effect of CS on the collagenolytic activities of 14 different human gingival fibroblast strains derived from healthy individuals with non‐inflammed gingiva was examined in vitro. There was marked heterogeneity among individuals in basal levels of collagenase activity, and there was also variation among the subpopulations derived from one strain. Fibroblasts from different individuals also varied markedly in their collagenolytic response to CS (0.1 to 0.75 μg/ml). In most strains, CS decreased collagenase activity, but in some, the drug caused no change or significantly increased activities. In most of the subpopulations CS significantly decreased collagenolytic activity.Two of the fibroblast strains and the subpopulations described above were examined for the production of immunoreactive collagenase and tissue inhibitor of metalloproteinase (TIMP). The two strains made similar amounts of collagenase, but differed markedly in TIMP levels; CS affected their collagenase production differently but had similar effects on TIMP. Among the subpopulations there was variation in the production of collagenase, although none made detectable levels of TIMP; they also varied in the production of both proteins in response to CS. In two of the subpopulations and in both strains at some concentrations, the effect of CS on the relative levels of collagenase and TIMP could account for the decreased collagenase activity; i.e., the level of collagenase was unchanged or decreased, and TIMP production was unchanged or increased.This study demonstrates the variation among individuals as well as intrastrain heterogeneity of human gingival fibroblasts with regard to collagenase activity and the production of collagenase and TIMP. The heterogeneity of the collagenolytic response of different gingival fibroblast strains and their subpopulations to CS treatment may partly explain the susceptibility of only some individuals to CS‐induced gingival enlargement.
Transcriptional element H4‐site II of cell cycle regulated human H4 histone genes is a multipartite protein/DNA interaction site for factors HiNF‐D, HiNF‐M, and HiNF‐P: Involvement of phosphorylation
Authors: van Wijnen Andre J. , Ramsey‐Ewing Anna L. , Bortell Rita , Owen Thomas A. , Lian Jane B. , Stein Janet L. , Stein Gary S. 
Published: May 2005

AbstractCell cycle regulated gene expression was studied by analyzing protein/DNA interactions occurring at the H4‐Site II transcriptional element of H4 histone genes using several approaches. We show that this key proximal promoter element interacts with at least three distinct sequence‐specific DNA binding activities, designated HiNF‐D, HiNF‐M, and HiNF‐P. HiNF‐D binds to an extended series of nucleotides, whereas HiNF‐M and HiNF‐P recognize sequences internal to the HiNF‐D binding domain. Gel retardation assays show that HiNF‐D and HiNF‐M each are represented by two distinct protein/DNA complexes involving the same DNA binding activity. These results suggest that these factors are subject to post‐translational modifications. Dephosphorylation experiments in vitro suggest that both electrophoretic mobility and DNA binding activities of HiNF‐D and HiNF‐M are sensitive to phosphatase activity. We deduce that these factors may require a basal level of phosphorylation for sequence specific binding to H4‐Site II and may represent phosphoproteins occurring in putative hyper‐ and hypo‐phosphorylated forms. Based on dramatic fluctuations in the ratio of the two distinct HiNF‐D species both during hepatic development and the cell cycle in normal diploid cells, we postulate that this modification of HiNF‐D is related to the cell cycle. However, in several tumor‐derived and transformed cell types the putative hyperphosphorylated form of HiNF‐D is constitutively present. These data suggest that deregulation of a phosphatase‐sensitive post‐translational modification required for HiNF‐D binding is a molecular event that reflects abrogation of a mechanism controlling cell proliferation. Thus, phosphorylation and dephosphosphorylation of histone promoter factors may provide a basis for modulation of protein/DNA interactions and H4 histone gene transcription during the cell cycle and at the onset of quiescence and differentiation.
Bellini, carpaccio, and receptors in the central nervous system
Authors: Merrill Jean E. 
Published: May 2005

AbstractWith the convergence of science from the fields of neurobiology and immunology, many exciting and challenging surprises have emerged regarding cytokines, neuroendocrine hormones, neuropeptides, excitatory amino acids, and their receptors. For some time neurobiologists have known that subsets of neural cells had different receptors for the same ligand. Those subsets of cells could be as different as neurons and astrocytes and as closely related as astrocytes from different lineages or anatomical areas. The neurobiological puzzle has been to determine the functional meaning of these differences. Immunologists in contrast have long understood the clear cut differences between T and B lymphocytes or T helper/inducer and T cytotoxic/suppressor cells and their response to cytokines. However, it is only very recently that they have discovered preferential use by these cells of different receptors for an identical cytokine ligand. Indeed, identical cytokines in the central nervous system and immune response may induce their pleiotropic responses by utilizing different receptors in these two systems. Immunologic paradigms may help neurobiologists predict the existence of subsets of neural cells and their function. Likewise, neurobiology may enable immunologists to predict roles for receptors in gene families as well as the existence of as yet unidentified receptors.
Purification and characterization of an osteoclast membrane glycoprotein with homology to manganese superoxide dismutase
Authors: Oursler Merry Jo , Li Ling , Osdoby Philip 
Published: May 2005

AbstractThe osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the inorganic and organic components of bone matrix. Isolated avian osteoclasts have been used to immunize mice and generate an osteoclast‐directed monoclonal antibody library (J. Cell Biology, 100:1592). A subset of these monoclonal antibodies recognizes antigens which are expressed on osteoclasts and which are absent or nearly so on multinucleated giant cells formed in vitro from monocyte or marrow mononuclear cells. One of these antibodies, designated 121F, has been used to identify and purify an osteoclast plasma membrane‐associated glycoprotein. Western blot analysis on disulfide bond‐reduced extracts from osteoclasts or multinucleated giant cells formed in vitro demonstrates that the 121F antibody recognizes a 150 kDa protein detectable only in osteoclasts. This high molecular weight protein has been purified by a combination of immunoaffinity and gel filtration chromatography procedures, in conjunction with electroelution of a single band from SDS‐polyacrylamide gels. Silver staining of the purified antigen on SDS‐polyacrylamide gels has revealed a single protein species larger than 200 kDa in its unreduced form and 150 kDa when disulfides are reduced. Isoelectric focusing of the purified antigen reveals a single species, having a neutral pl point of 6.95. Whereas endoglycosidase treatment and lectin affinity chromatographic analyses demonstrate that the antigen recognized by the 121F antibody possesses complex N‐linked sugars, trifluoromethanesulfonic acid treatment indicates there are no additional O‐linked carbohydrate components. Periodate oxidation and monosaccharide hapten inhibition studies provide no evidence for the antigenic epitope bound by the 121F antibody being carbohydrate in nature. Although the native antigen is blocked at its N‐terminus, amino acid analysis of a hydroxylamine generated peptide disclosed a striking relationship between the osteoclast antigen recognized by the 121F monoclonal antibody and manganese and iron superoxide dismutase. Therefore, in addition to serving as a distinguishing cell type–specific marker for osteoclasts, this cell surface glycoprotein may function directly in osteoclast‐mediated bone resorption.
Buffering of intracellular calcium in response to increased extracellular levels in mortal, immortal, and transformed human breast epithelial cells
Authors: Ochieng Josiah , Tahin Quivo S. , Booth Charles C. , Russo Jose 
Published: May 2005

AbstractExtracellular levels of calcium at 1.05 mM or higher induce terminal differentiation and senescence in the mortal (MCF‐10M) line of human breast epithelial cells, but does not retard the growth or induce differentiation in the immortal (MCF‐10A) and oncogene transformed (MCF‐10AneoT) lines. Intracellular levels of calcium and inositol triphosphate were determined in MCF‐10M, MCF‐10A, and MCF‐10AneoT, under conditions of low and high extracellular calcium. We hereby report that increases in extracellular calcium is translated into significant increases in intracellular levels of calcium and inositol triphosphate in MCF‐10M, but not in MCF‐10A and MCF‐10AneoT. This difference in the apparent calcium buffering capacity between the mortal and the immortalized human breast epithelial cells could account for the latter's unperturbed growth potential in high extracellular calcium environment.
The effect of a phorbol ester on the lipid microviscosity of two endoplasmic reticulum membrane fractions isolated from krebs II ascites cells
Authors: Maltseva E. L. , Palmina N. P. , Pryme I. F. 
Published: May 2005

AbstractThis paper deals with microviscosity parameters and thermoinduced structural transitions in the lipids of smooth and heavy rough endoplasmic reticulum membranes isolated from Krebs II ascites cells incubated with the phorbol ester 12–0‐tetradecanoyl‐phorbol‐13‐acetate. The phorbol ester was found to bring about a threefold increase in the microviscosity of the lipids in heavy rough membranes. Spin probe I (2,2,6,6‐tetrahydro‐4‐capryloyl‐oxypiperidine‐1‐oxyl), localized in the surface layer of the membrane lipids, gave results which indicate an increased number of thermoinduced structural transitions in the smooth membranes in the treated cells due to the transitions occurring at relatively low temperature and a decreased number of such transitions in the heavy rough fraction especially at high temperature. For 5,6‐benzo‐2,2,4,4‐tetramethyl‐1,2,3,4‐tetrahydro‐γ‐carboline‐oxyl, probe II, mainly distributed in the annular lipids, a decrease in the number of low temperature transitions in the smooth fraction was observed, while an increase occurred in the heavy rough one. The results obtained are discussed in terms of the effect of phorbol esters as promoters of tumor progression.