bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced shear stress: A major component in the ability of mammalian tissues to form three‐dimensional assemblies in simulated microgravity
Authors: Goodwin T. J. , Prewett T. L. , Wolf David A. , Spaulding G. F.
Published: May 2005

BHK‐21 cells were cultured under various shear stress conditions in an Integrated Rotating‐Wall Vessel (IRWV). Shear ranged from 0.5 dyn/cm2 (simulated microgravity) to 0.92 dyn/cm2. Under simulated microgravity conditions, BHK‐21 cells complexed into three‐dimensional cellular aggregates attaining 6 × 106 cells/ml as compared to growth under 0.92 dyn cm2 conditions. Glucose utilization in simulated microgravity was reduced significantly, and cellular damage at the microcarrier surface was kept to a minimum. Thus, the integrated rotating wall vessel provides a quiescent environment for the culture of mammalian cells. © 1993 Wiley‐Liss, Inc.
DNA repair and repair fidelity in metastatic variants of the B16 murine melanoma
Authors: Usmani B. A. , Lunec J. , Sherbet G. V.
Published: May 2005

We have examined the concept of genomic instability in relation to the metastatic progression of low (F1) and high metastasis (BL6, ML8) clones of the B16 mouse melanoma, by using a mutation assay, and DNA strand break repair and repair fidelity assays. The frequency of induced ouabain resistant colonies between the variant cell lines was consistent with the difference between their metastatic properties. Survival data for X‐irradiation and bleomycin were similar among the 3 cell lines. When X‐rays or bleomycin were used to induce strand breakage, no difference was detectable in either the rate or extent of DNA repair using the techniques of alkaline unwinding and alkaline elution for total strand breaks, and neutral elution for double strand breaks. DNA repair fidelity was measured using the PMH16 plasmid. A Kpn I restriction site was used to introduce a break within the gpt gene of the plasmid, prior to transfection. We found that ∼ 100% and ∼ 65% of the highly metastatic ML8 and BL6 clones, respectively, religated the gene with the required fidelity, compared with only ∼ 25% of the low metastasis F1 clones. In summary, the metastatic variants show similar sensitivities to X‐irradiation and bleomycin, but a differential response to EMS. This difference is not reflected in any subsequent DNA strand break religation, but the variants do differ in their fidelity of repair. However, although the fidelity of DNA religation is related to metastatic potential, it is not consistent with the mutation frequency data. © 1993 Wiley‐Liss, Inc.
Drosophila acidic ribosomal protein rpA2: Sequence and characterization
Authors: Olson Pamela F. , Salo Tuula , Garrison Katherine , Fessler John H.
Published: May 2005

A cDNA encoding the Drosophila melanogaster acidic ribosomal protein rpA2 was cloned and sequenced. rpA2 is homologous to the Artemia salina acidic ribosomal protein eL12′. In situ hybridization to salivary gland polytene chromosomes localizes the rpA2 gene to band 21C. It is a single copy gene, with an mRNA of 0.8 kb. Two‐dimensional gel electrophoresis of Drosophila ribosomal proteins followed by immuno‐blotting showed that the rpA2 protein has an apparent relative mobility in SDS of 17 kD and an isoelectric point less than pH 5.0. Although the Drosophila gene rp21C may be the same as rpA2, the reported sequences differ. Comparisons of the aligned nucleotide sequences coding for the acidic ribosomal proteins rpA1 and rpA2 of Drosophila with those of other eukaryotes support the view of two separate, though closely related, groups of acidic proteins. Comparison with the Artemia homologues suggests that nucleotide identity may have been conserved by some constraint that acts in addition to the requirement for substantial similarity of amino acid sequences. © 1993 Wiley‐Liss, Inc.
High mannose type N‐linked oligosaccharides on endothelial cells may influence β2 integrin mediated neutrophil adherence in vitro
Authors: Sriramarao P. , Berger Elaine , Chambers J. David , Arfors Karl‐E. , Gehlsen Kurt R.
Published: May 2005

We report herein on the role of N‐linked oligosaccharide processing of endothelial cell surface proteins on the adhesion of neutrophils. Monolayers of human umbilical vein endothelial cells were treated for 24 h with deoxymannojirimycin (DMJ), an inhibitor of golgi mannosidase I, which results in changes in glycoprotein processing, and then incubated with neutrophils to examine their ability to adhere to the treated endothelial cells. Treatment with DMJ, which leads to accumulation of high mannose type oligosaccharides, resulted in a twofold increase in adherence of phorbol ester (PMA) activated neutrophils compared to attachment to untreated endothelial cells. This adherence was likely mediated by the β2 integrin, Mac‐1, and could be specifically inhibited with monoclonal antibodies to ICAM‐1 and to the integrin β2 subunit. Similarly, IL‐1 treatment resulted in a β2 integrin mediated increase in neutrophil adherence to the DMJ treated endothelial cells in a dose dependent manner. However, the IL‐1 induced adherence was not significantly inhibited by the anti‐ICAM‐1 antibody, thus, suggesting the presence of other inducible components on the endothelial cell surface. Our results demonstrate that alterations in glycosylation of N‐linked oligosaccharides, resulting in the synthesis of high mannose type sugars on molecules that may interact with the β2 integrins, leads to an increased adherence of PMA activated neutrophils to endothelial cells. © 1993 Wiley‐Liss, Inc.
Enhanced phosphorylation of a 65 kDa protein is associated with rapid induction of stress proteins in 9L rat brain tumor cells
Authors: Lai Yiu‐Kay , Shen Chi‐Hsiu , Cheng Ting‐Jen , Hou Ming‐Ching , Lee Wen‐Chuan
Published: May 2005

Induction of heat‐shock proteins and glucose‐regulated proteins in 9L rat brain tumor cells can be differentially elicited by sodium arsenite, cadmium chloride, zinc chloride, copper sulfate, sodium fluoride, and L‐azetidine‐2‐carboxylic acid. The kinds of stress protein induced by the above chemicals varied considerably, mainly determined by the nature and the concentration of the chemicals, as well as the treatment protocols. In addition, at the concentrations where stress proteins can be induced, the above chemicals were able to suppress general protein synthesis and were cytotoxic. Enhanced phosphorylation of a protein with an apparent molecular weight of 65 kDa was detected during the induction of stress proteins except in azetidine treatments during which uptake of phosphate by the cells was impaired after prolonged incubation. The phosphate moiety on the 65 kDa phosphoprotein appeared to be alkaline‐stable and two‐dimensional gel electrophoresis revealed that the phosphoprotein resolved into four isoforms with isoelectric points ranging from 5.1 to 5.6. Enhanced phosphorylation of the same protein was also detected in heat‐shocked and withangulatin A‐treated 9L cells in which stress proteins were induced. It is suggested that this phosphoprotein may be a common target for heat stress response‐stimulated phosphorylation and important in the further metabolic responses of the cell to stress. © 1993 Wiley‐Liss, Inc.
Protein kinase C: Mediator or inhibitor of insulin action?
Authors: Considine R. V. , Caro J. F.
Published: May 2005

The role of protein kinase C in insulin signal transduction i s controversial It has been postulated that protein kinase C i s activated by insulin and that the kinase i s directly involved in insulin‐mediated metabolic processes. In opposition to this view i s the hypothesis that protein kinase C is not activated by insulin and, more importantly, may be responsible for attenuation of the insulin signal The evidence for and against protein kinase C as a mediator of the insulin signal will be put in perspective followed by discussion of the possible role of the kinase in the pathogenesis of insulin resistance in type II diabetes.
Therapeutic effects of electromagnetic fields in the stimulation of connective tissue repair
Authors: Aaron Roy K. , Ciombor Deborah McK.
Published: May 2005

The therapeutic effects of electric and magnetic fields have been studied largely for their promotion of connective tissue repair. The most widely studied application concerns bone repair and deals with acceleration of the healing of fresh fractures, delayed and non‐unions, incorporation of bone grafts, osteoporosis, and osteonecrosis. More recently the effects of these fields upon the repair of cartilage and soft fibrous tissues have been described. In all these experimental systems and clinical applications an acceleration of extracellular matrix synthesis and tissue healing has been observed. A degree of specificity, in terms of the parameters of applied energy and biological response, is hypothesized.
Regulation of prolactin receptor expression by the tumour promoting phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate in human breast cancer cells
Authors: Ormandy Christopher J. , Lee Christine S. L. , Kelly Paul A. , Sutherland Robert L.
Published: May 2005

In both the normal and malignant human breast, cellular sensitivity to the proliferative and differentiative activities of the lactogenic hormones is conferred by expression of the prolactin receptor (PRLR). The PRLR is regulated by steroid hormones; however, recent findings have suggested that PRLR may also be regulated by protein kinase C. To examine this possibility we have studied the effect of various modulators of PKC activity on PRLR binding activity and gene expression in five PRLR positive human breast cancer cell lines. Treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a tumour promoter and modulator of PKC activity, decreased PRLR binding activity in all cell lines examined. In MCF‐7 cells, 10 nM TPA caused a 70% loss of PRLR mRNA after 12 h, paralleled 3 h later by a comparable loss of cell surface PRLR. Mezerein, a non‐phorbol ester modulator of PKC activity and 1,2‐dioctanoyl‐sn‐glycerol, a permeant analogue of the endogenous activator of PKC, also reduced PRLR binding activity, and gene expression in a time‐ and concentration‐dependent manner. Cycloheximide failed to abrogate the TPA‐induced decline in PRLR mRNA levels, indicating that this process was not dependent upon continuing protein synthesis. No change in the stability of PRLR mRNA was observed during 24 h of TPA treatment and TPA reduced the rate of PRLR gene transcription within 3 h of treatment. These results demonstrate that modulators of PKC activity reduce PRLR binding activity and gene expression, implicating this signal transduction pathway in PRLR regulation.
Alterations of collagen mRNA expression during retinoic acid induced chondrocyte modulation: Absence of untranslated α1(I) mRNA in hyaline chondrocytes
Authors: Dietz Uwe , Aigner Thomas , Bertling Wolf M. , von der Mark Klaus
Published: May 2005

Retinoic acid (RA) has been shown to rapidly modulate the collagen expression pattern of chondrocytes in vitro at doses of 1–10 μM. Embryonic chicken sternal chondrocytes stop synthesizing the cartilage‐specific type II collagen within 2–4 days of RA treatment and turn on the synthesis of types I and III collagen and fibronectin. While suppression of type II collagen synthesis and onset of type III collagen and fibronectin synthesis have been shown to be regulated at the transcriptional level, conflicting data are available on a possible post‐translational regulation of α1(I) collagen gene expression. In this study we demonstrate by comparing a commonly used α1(I) cDNA probe from the 3′ end of the α1(I) mRNA with a newly prepared α1(I) specific cDNA probe from the 5′ end (p1E1) that—in contrast to previous reports—chicken sternal chondrocytes do not contain untranslated α1(I) mRNA which may become translatable after RA treatment. By in situ hybridization we show the absence of cytoplasmic α1(I) mRNA from chondrocytes and its presence in the perichondrium of sternal cartilage. Perichondral cells might have contaminated sternal chondrocyte preparations, explaining low levels of α1(I) mRNA seen by Northern hybridization and RNase protection assays of chicken sternal cartilage mRNA even with the p1E1 probe. We show by Northern hybridization and metabolic labeling with 3H‐proline followed by SDS‐gel electrophoresis that retinoic acid at 3 μM suppresses type II, IX, and X collagen gene expression within 2 days both at the mRNA and protein level and induces the onset of α1(I), α2(I), and α1(III) expression within 3 days. No expression of CRABP, the cellular retinoic acid binding protein, was seen in RA‐treated or control chondrocytes, indicating that CRABP protein is not involved in the RA‐induced modulation of the chondrocytes.
Cell cycle dependent distribution of proliferating cell nuclear antigen/cyclin and cdc2‐kinase in mouse T‐lymphoma cells
Authors: Brott David A. , Alvey James D. , Bleavins Michael R. , de la Iglesia Felix A. , Lalwani Narendra D.
Published: May 2005

The aim of the present study was to investigate bromodeoxyuridine (BrdU) uptake and coordinated distribution of proliferating cell nuclear antigen (PCNA) and p34‐cdc2‐kinase, two important proteins involved in cell cycle regulation and progression. Flow cytometric analysis of marker proteins in freshly plated mouse T‐lymphoma cells (Yac‐1 cells), using fluorescein isothiocyanate (FITC)‐labeled specific antibodies, showed PCNA distributed throughout the cell cycle with increased intensity in S‐phase. PCNA is essential for cells to cycle through S‐phase and its synthesis is initiated during late G1‐phase before incorporation of BrdU and remains high during active DNA replication. The intensity of PCNA fluorescence increases with the duration of incubation after plating. The cdc2‐kinase was detectable in all phases of the cell cycle and the G2‐M‐phase appears to have the maximum concentrations. The cell cycle analysis of high dose colcemid (2 μg/ml) treated Yac‐1 cells showed an aneuploid or hypodiploid population. Although the G2‐M‐phase seems to be the dominating population in aneuploid cells, the concentrations of cdc2‐kinase were variable in this phase of cell cycle. The colcemid treatment at 25 ng/ml arrested 96% of cells in S‐phase and G2‐M‐phase, but PCNA expression was evident in a portion of the cell population in G2‐M‐phase. Although cells blocked in M‐phase seem to have high levels of cdc2‐kinase, colcemid renders them inactive. From these data, it appears that the down regulation and/or inactivation of cdc2‐kinase could be responsible for the colcemid arrest of cells in M‐phase.
Association of a human H1 histone gene with an H2A pseudogene and genes encoding H2B.1 and H3.1 histones
Authors: Kardalinou Efterpi , Eick Stefan , Albig Werner , Doenecke Detlef
Published: May 2005

A cluster of human histone genes was found on three overlapping clones isolated from cosmid and bacteriophage libraries. These three overlapping segments of the human genome comprise genes coding for H3.1, an H2A pseudogene, and an H2B.1 gene downstream of the previously characterized H1.2 gene. The cosmid clone covers 30 kb upstream of the H1.2 gene and overlaps with two phage clones covering the core histone genes and the pseudogene. The same arrangement of an H3 gene, an H2A pseudogene and an H2B gene downstream of an H1 gene has been described within a mouse histone gene cluster [Yang et al.: J Biol Chem 262:17118–17125, 1987; Gruber et al.: Gene 95:303–304, 1990].
Endothelial cells exhibiting angiogenesis in vitro proliferate in response to TGF‐β1
Authors: Iruela‐Arispe M. Luisa , Sage E. Helene
Published: May 2005

Transforming growth factor‐β1 (TGF‐β1) has been implicated in the positive regulation of angiogenesis in vivo, whereas it inhibits the proliferation of endothelial cells in vitro. To reconcile these apparently contradictory effects, we have investigated the effect of TGF‐β1 on bovine aortic endothelial cells that exhibit spontaneous angiogenesis in vitro. We show that concentrations of TGF‐β1 which stimulate proliferation of cells that form endothelial cords and/or tubes inhibit proliferation of the same cells grown at subconfluent densities. An increase in cell number of 35% over control cultures was achieved with 0.5 ng TGF‐β1/ml. The proliferative effect was blocked by antibodies against TGF‐β. Immunological detection of BrdU‐labeled nuclei revealed an increase greater than 220% in cells treated with TGF‐β1. Moreover, a population of cells within the cords appeared to be a selective target for this cytokine. The stimulatory effect was not restricted to bovine aortic endothelial cells, as similar results were obtained with endothelial cells derived from rat microvessels. Significant levels of active TGF‐β1 were detected in cultures containing cords/tubes, whereas only latent TGF‐β1 was detected in subconfluent cultures. We show further that endothelial cells exhibiting angiogenesis in vitro secrete plasminogen activator, an enzyme that regulates activation of TGF‐β. The major increases in mRNA transcripts for extracellular matrix proteins that are typically associated with TGF‐β1 were not seen in cells exhibiting angiogenesis in vitro. Since the formation of tubular networks requires both invasion and proliferation, we propose that TGF‐β1 is a major morphoregulatory factor in angiogenesis that specifically controls endothelial cell proliferation and extracellular matrix turnover.
Studies of the Levamisole inhibitory effect on rat stromal‐cell commitment to mineralization
Authors: Klein Benjamin Y. , Gal Irena , Segal David
Published: May 2005

The ability of Levamisole to decrease mineralization in skeletal tissue is usually related to its effect on alkaline phosphatase (ALP). However, Levamisole is also suspected to diminish mineralization by an additional mechanism which is unrelated to the ALP control of apatite crystal growth. To delineate the time in differentiation during which Levamisole inhibits mineralization, a tissue culture model system of bone marrow stromal cells was used. Secondary cultures of stromal cells were propagated in osteoprogenitor cell (OPC) induction medium for three weeks, followed by measurement of calcium precipitation. In situ ALP assays at pH 7.6 were also performed. When cells were cultured with 0.2 mM Levamisole for three weeks, Day 20 values of calcium precipitates were lower than in controls, but Day 20 ALP values were paradoxically higher. The correlation between calcium and ALP within each group was low. The correlation slightly improved, in uninhibited cultures, when Day 21 calcium values were matched with earlier Day 12 ALP values. This suggested the existence of a Levamisole‐sensitive mechanism for mineralization inhibition effective prior to the culture's mineralization stage. To focus on this early effect on mineralization Levamisole was added to stromal cultures on different days and removed on Day 12. Levamisole decreased Day 21 mineralization when added on Days 0, 3, 5, and 7, but not when added on Day 9. The Levamisole‐induced inhibition of mineralization was accompanied by an increase in Day 12 ALP specific activity, compared to controls, when added from Day 5 and thereafter. The results indicate that part of the ability of stromal cells to mineralize is determined during the first week of culture. The early inhibitory effect of Levamisole on mineralization was associated with increased Day 12 ALP activity.
Expression and localization of elements of the plasminogen activation system in benign breast disease and breast cancers
Authors: Jankun Jerzy , Merrick Hollis W. , Goldblatt Peter J.
Published: May 2005

The malignant potential of solid tumors is related to the ability to invade adjacent tissue and to metastasize. These properties of cancer cells depend on the synthesis of proteolytic enzymes which are able to digest adjacent connective tissue and basement membranes. We hypothesized that all elements of the plasminogen activation system might be overexpressed in malignant human breast tumors, functioning as an essential element in tumor invasion and metastasis. As determined by histopathological methods, the malignant tumors showed statistically significantly higher expression of urokinase plasminogen activator (uPA), type‐1 plasminogen activator inhibitor (PAI‐1), and especially urokinase plasminogen activator receptor (uPAR) than benign tissues. All those elements were present in higher amounts in the cancer cells than in the cells of benign or normal breast tissues. High exhibition of tissue plasminogen activator (tPA) found in cancer seems to be random and not related to the malignant or benign state, since benign and malignant tumors show overexpression of tissue plasminogen activator with similar frequency. When the tumors express high amounts of uPA, they express a high amount of uPAR in 50% of cases and PAI‐1 in 57.3% of cases. When urokinase is expressed in low amount, the receptor is low in 28.6% and inhibitor in 21.4% of malignant breast tumors. This statistically significant consensus, 78.6% in the case of urokinase and its receptor and 78.6% in case of urokinase and its inhibitor, suggests that these activities may be the result of a unique mechanism of control, activated in the last steps of malignant transformation.
Endothelin modulates osteopontin and osteocalcin messenger ribonucleic acid expression in rat osteoblastic osteosarcoma cells
Authors: Shioide Mitsuhiro , Noda Masaki
Published: May 2005

Endothelins (ETs) are vasoconstrictive peptides produced mainly by endothelial cells. The ET receptors are expressed in many types of cells including osteoblast‐like cells. The purpose of this study was to examine the effects of endothelin on the expression of osteoblastic phenotype‐related genes. We found that endothelin‐1 (ET‐1) enhanced approximately two‐fold the mRNA expression of both osteopontin and osteocalcin genes in rat osteoblastic osteosarcoma ROS17/2.8 cells. These effects were dose‐dependent, peaking at 10−7 M. The ET‐1 enhancement of the abundance of osteopontin and osteocalcin mRNAs was time‐dependent, with a maximal effect at 24 h. ET‐1 modulation of the expression of the two phenotype‐related gene products of osteoblasts suggests that endothelin is one of the cytokines which modulate osteoblastic functions and that this molecule may play a role in the regulation of bone metabolism.
Purification and characterization of NADPH‐cytochrome P‐450 reductase from rat epidermis
Authors: Takahara Hidenari , Zaidi Syed I. A. , Mukhtar Hasan , Handa Masafumi , Epstein William L. , Fukuyama Kimie
Published: May 2005

NADPH‐cytochrome P‐450 oxidoreductase (P‐450 red) transfers reducing equivalents from NADPH to cytochrome P‐450 (P‐450) in the monooxygenase system. Detergent solubilized proteins from the membrane fraction of neonatal rat epidermis were purified by 2′,5′‐ADP‐agarose affinity column chromatography. The purified protein showed an apparent homogeneity on sodium dodecylsulfate‐polyacrylamide gel electrophoresis and molecular weight was estimated to be 78 kDa. NADPH‐cytochrome c reductase activity increased by 95‐fold in the purified enzyme. Epidermal P‐450 red in vitro reconstituted benzo(a)pyrene hydroxylase activity in a dose dependent manner with P‐450 purified from either rat liver or epidermis. Western blot analysis demonstrated that epidermal P‐450 red immunologically cross reacts to liver P‐450 red. Immunohistochemical staining showed that the enzyme was predominantly localized in the epidermis. The intensity of immunohistochemical staining of rat skin sections and tissue distribution did not change in the skin treated with β‐naphtoflavone, which results in a substantial increase in P‐450 1A1 activity. Quantitative assessment of P‐450 red in treated and untreated epidermis also showed no change. These findings indicate that constitutive P‐450 red, fully capable of supporting P‐450, exists in rat epidermis, and can function in metabolism of endogenous and exogenous compounds.
1α,25‐Dihydroxyvitamin D3‐induced changes in intracellular pH in osteoblast‐like cells modulate gene expression
Authors: Jenis Louis G. , Lian Jane B. , Stein Gary S. , Baran Daniel T.
Published: May 2005

1α,25‐Dihydroxyvitamin D3 exerts rapid nongenomic effects on rat osteoblast‐like cells independent of the classic nuclear receptor. These effects include changes in phospholipid metabolism and cell calcium. Intracellular calcium itself has been proposed to regulate intracellular pH in osteoblast cell lines. The purpose of this study was to determine the effect of 1α,25‐dihydroxyvitamin D3 on intracellular pH, the relationship of changes in calcium to changes in pH, and the role of pH changes in genomic activation. 1α,25‐Dihydroxyvitamin D3 increased intracellular pH within 10 min in rat osteoblast‐like cells, an effect that was inhibited by removal of extracellular sodium and by the biologically inactive epimer 1β,25‐dihydroxyvitamin D3. The hormone increased intracellular calcium in Quin 2 loaded cells in the presence and absence of extracellular sodium. The 1α,25‐dihydroxyvitamin D3‐induced increments in osteocalcin and osteopontin mRNA levels were abolished in sodium‐free medium. The results indicate that 1α,25‐dihydroxyvitamin D3‐induced increments in cellular calcium precede cell alkalinization and that these changes in intracellular pH may modulate steady‐state mRNA levels of genes induced by vitamin D.
Extracellular annexin II is associated with divalent cation‐dependent tumor cell–endothelial cell adhesion of metastatic RAW117 large‐cell lymphoma cells
Authors: Tressler Robert J. , Updyke Timothy V. , Yeatman Timothy , Nicolson Garth L.
Published: May 2005

Using fixed microvessel endothelial cell monolayers the molecules involved in the adhesion of liver‐preferring murine RAW117 large cell lymphoma cells to murine liver‐derived microvessel endothelial cells were identified by affinity isolation. Detergent lysates obtained from poorly (P) or highly (H10) liver‐metastatic cells inhibited RAW117‐H10 cell adhesion to hepatic sinusoidal endothelial (HSE) cell monolayers. Allowing detergent lysates of cell surface‐labeled RAW117 cells to bind to fixed HSE cell monolayers and eluting the bound components indicated that several tumor cell surface molecules ( ∼ 70, ∼ 35, ∼ 32, ∼ 22, and ∼ 14 kDa) might be involved in RAW117 cell‐HSE cell adhesion. The ∼ 35 kDa component was cation dependent in its binding to target HSE cells. Increasing detergent concentration had no effect on binding of the ∼ 35 kDa component to HSE cell monolayers, whereas treatment with 0.5 M NaCl resulted in its selective elution from HSE cells. Incubation of the HSE cell monolayers with detergent lysates from cell surface‐labeled RAW117‐H10 cells resulted in selective depletion of the ∼ 35 kDa component, suggesting that the binding is saturable. This divalent cation‐dependent molecule is one of the major tumor cell surface components bound by several types of endothelial cells and murine hepatocytes, whereas there was poor binding of this component to unfixed or fixed human red blood cells. The purified, partially ( ∼ 40%) sequenced molecule had amino acid sequence identity with murine but not bovine annexin II, indicating that it was not bound from the bovine serum used to grow RAW117 cells. Using antibodies specific for annexin II flow cytometery indicated equivalent amounts of annexin II are expressed on RAW117 cell surfaces in the absence or presence of excess EDTA, whereas annexin I was only found in low amounts on the surfaces of RAW117 cells. Annexin II antibodies inhibited by ∼ 40–50% the adhesion of RAW117 tumor cells to live or fixed endothelial cells, and purified tumor cell surface fractions containing the ∼ 35 kDa component partially inhibited ( ∼ 35%) RAW117 cell–HSE cell adhesion. The data indicate that annexin II is expressed on the extracellular surface of RAW117 cells, and cell surface–annexin II mediates a portion of the Ca2+‐dependent RAW117 cell adhesion to liver microvessel endothelial cells.
Activities of growth factors in preimplantation embryos
Authors: Adamson Eileen D.
Published: May 2005

The development of the mammalian preimplantation embryo in vitro occurs more slowly and less successfully compared to development in the uterus. The fact that it can occur at all in a defined protein‐free medium suggests that the process is autonomous. Accumulated evidence indicates that a number of peptide growth factors contribute in an autocrine fashion to preimplantation development. Other growth factors are maternally derived and act in a paracrine manner on the embryo. Some of these factors such as insulin‐related factors stimulate growth preferentially, but others such as epidermal growth factor (EGF) play more important roles in differentiation. Several cytokines appear to be implicated in peri‐implantation events and in maternal‐fetal interactions. At this stage, the data are mostly descriptive. Are all these different growth factors and receptors necessary for early development? Some implications of apparent redundancy of gene expression are discussed and future studies are predicted.
Hypothalamic releasing hormones mediating the effects of interleukin‐1 on sleep
Authors: Payne Linda C. , Obál Ferenc , Krueger James M.
Published: May 2005

There is a substantial literature describing the interactions between the endocrine and immune systems. Although such interactions are less well known within the brain, one major brain function altered during inflammation and infection and by several endocrine hormones is sleep. Pathological disturbances, be they inflammation, infectious disease, and/or sleep deprivation, result in altered hypothalamus‐pituitary function and cytokine metabolism. In respect to hormone secretion from the pituitary, cytokines are now recognized to play an important role in modulating the neuroendocrine system. Changes in sleep provide a useful illustration of the interactions between cytokines and the hypothalamus‐pituitary axis. Evidence linking interleukin‐1 (IL‐1) to growth hormone releasing hormone and to corticotropin releasing hormone in regard to their effects on sleep is reviewed.
Regulation of angiotensin I‐converting enzyme in cultured bovine bronchial epithelial cells
Authors: Müns Günther , Vishwanatha Jamboor K. , Rubinstein Israel
Published: May 2005

The purpose of this study was to determine whether angiotensin I‐converting enzyme (ACE) is present in cultured bovine bronchial epithelial cells (BBECs) and whether its activity can be modulated. We found that extracts of confluent monolayers of cultured BBECs degraded [glycine‐1‐14C]hippuryl‐L‐histidyl‐L‐leucine at a rate of 843 ± 66 pmol/hr/mg protein (mean ± SEM, n = 5). In addition, we found that the enzyme was shed into the culture medium. ACE activity in BBECs was inhibited by three selective, but structurally different, ACE inhibitors (captopril, quinapril, and cisalaprilat) with an IC50 of approximately 2 nM. Increasing chloride concentration in the assay buffer resulted in an increase in BBECs ACE activity of 63%. Enzyme activity was also modulated by the presence of zinc cation in the assay buffer. Addition of dexamethasone to the culture medium was associated with a significant increase in BBECs ACE activity (P < 0.05), which was inhibited by the steroid receptor antagonist RU 38486. Western blot analysis of BBECs, tracheal and bronchial mucosal strips utilizing a cross‐reacting rabbit anti‐mouse ACE antibody, showed a faint 175 kDa band and additional strong 52 kDa and 47 kDa band. The mechanism of generation of the low M.W. bands is unknown. Our data indicate the presence of ACE in cultured BBECs and that enzyme activity can be modulated.
Further purification and characterization of a multienzyme complex for DNA synthesis in human cells
Authors: Li Congjun , Cao Long‐Guang , Wang Yu‐Li , Baril Earl F.
Published: May 2005

The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract‐post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362–6374] was purified by poly (ethylene glycol) precipitation, Q‐Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase α‐primase, a 3′,5′ exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase α,β and DNA ligase I, showed that polymerase α and DNA ligase I are localized to granular‐like foci within the nucleus during S‐phase. In contrast, DNA polymerase β, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.
Catecholamine stimulated lipolysis in differentiated human preadipocytes in a serum‐free, defined medium
Authors: van de Venter Maryna , Litthauer Derek , Oelofsen Willem
Published: May 2005

Adipocyte precursors from the stromal vascular fraction of human adipose tissue were allowed to differentiate in serum‐free defined medium, whereafter their catecholamine stimulated lipolytic response was compared to that of mature isolated human adipocytes. Seventy‐five to ninety percent of the fibroblast‐like cells accumulated lipid droplets and glycerol‐3‐phosphate dehydrogenase activities of 1,000–2,800 mU/mg protein were measured in cell homogenates of differentiated cells. Lipolysis could be stimulated by both isoproterenol and norepinephrine in both differentiated preadipocytes as well as mature adipocytes. The results obtained with β‐adrenergic agents suggested the presence of a higher affinity receptor in differentiated preadipocytes as compared to mature adipocytes. Mature adipocytes responded well to β‐adrenergic agents, but no antilipolytic α2‐adrenergic response was observed in the differentiated preadipocytes. The presence of Gi proteins in the differentiated preadipocytes was suggested by the antilipolytic effect of adenosine as well as the lipolytic activity generated by pertussis toxin. In conclusion, our medium supported the differentiation of a very high percentage of human preadipocytes which developed a sensitive β‐adrenergic lipolytic response but which lacked an α2‐adrenergic antilipolytic response.
A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro
Authors: Wu Yan , Hickey Robert , Lawlor Kenneth , Wills Philip , Yu Fang , Ozer Harvey , Starr Robyn , Quan Jiang Yuan , Lee Marietta , Malkas Linda
Published: May 2005

We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion‐exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases α and δ, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52‐cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T‐antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co‐purify with the complex.
c‐myc Gene expression is localized to the myocyte following hemodynamic overload in vivo
Authors: Pollack Pia S. , Houser Steven R. , Budjak Ricardo , Goldman Bruce
Published: May 2005

Expression of the proto‐oncogene c‐myc increases in the hemodynamically overloaded heart, but expression by cardiac myocytes has not been shown. To address this issue, right ventricular overload was induced in cats by pulmonary artery banding. Expression of c‐myc and α‐skeletal actin mRNA were determined by Northern analysis. Immuno‐reactive Myc protein was identified by histochemical staining.

Steady state levels of c‐myc mRNA peaked within 2 h after banding. Levels of α‐skeletal actin mRNA were maximally increased 48 h–1 week after banding and were still elevated at 1 month. Prominent staining of myocyte nuclei for immunoreactive Myc protein was detected 48 h after banding although a few interstitial nuclei were also positive.

These studies show that c‐myc and α‐skeletal actin gene expression are upregulated in a large animal model of hemodynamic overload. The localization of the immunoreactive Myc protein to right ventricular myocyte nuclei after pulmonary artery banding supports the hypothesis that c‐myc induction is part of a general response in cardiac hypertrophy that is common to many mammalian species.
Transient increase in vimentin phosphorylation and vimentin‐HSC70 association in 9L rat brain tumor cells experiencing heat‐shock
Authors: Cheng Ting‐Jen , Lai Yiu‐Kay
Published: May 2005

Characteristic changes in vimentin were studied in 9L rat brain tumor cells treated at 45°C. During heat‐shock treatment, vimentin molecules were rapidly phosphorylated and reorganized from a filamentous form into a perinuclear higher‐order structure that was less extractable by nonionic detergent. These effects were found to be highly transient, peaked at 30 min after the onset of heat‐shock treatment, and subsided thereafter. Simultaneously, the solubility of the constitutively expressed heat‐shock protein70 (HSC70) was also temporarily decreased and the kinetics was identical to that of vimentin. The results indicated that HSC70 and vimentin were co‐insolubilized during the heat‐shock treatment. We propose that the reorganization of the intermediate filaments resulted from enhanced phosphorylation of vimentin leads to the concurrent association of HSC70 to the intermediate filaments. This process may play an essential role in regulating heat‐shock genes.
TGFβ1 Prevents the down‐regulation of type I procollagen, fibronectin, and TGFβ1 gene expression associated with 3T3‐L1 pre‐adipocyte differentiation
Authors: Bortell Rita , Owen Thomas A. , Ignotz Ronald , Stein Gary S. , Stein Janet L.
Published: May 2005

Pre‐adipocyte 3T3‐L1 cells, after an appropriate induction stimulus, proceed through a defined change in morphology as differentiation progresses. Transforming growth factor β1 (TGFβ1) is able to block the morphological and biochemical changes which occur with differentiation of these cells if given within 36–40 h of induction [Ignotz and Massague (1985): Proc Natl Acad Sci USA 82:8530–8534]. To begin to elucidate the role of the extracellular matrix in adipogenesis, as well as the mechanism whereby TGFβ1 inhibits differentiation, we examined the expression of two extracellular matrix genes, type I (α1) procollagen and fibronectin, as well as endogenous TGFβ1. Confluent cells were induced to differentiate by treatment with insulin, dexamethasone, and isobutylmethylxanthine in the presence or absence of TGFβ1. Following 6 days of treatment, the cells in the differentiated group acquired the rounded shape of mature adipocytes; the cytosol of these cells also contained numerous lipid‐filled vesicles, as demonstrated by oil red O staining. Cells treated with the differentiation compounds in the presence of TGFβ1 maintained the fibroblast‐like appearance of control cells and did not stain with oil red O. At the level of gene expression, both procollagen and fibronectin mRNAs were down‐regulated during differentiation of 3T3‐L1 cells. When cells from the control or differentiation groups were treated with TGFβ1, there was a 2–5‐fold induction of procollagen and fibronectin mRNAs throughout the 6‐day time course. No change in type I procollagen transcription was observed by nuclear run‐on analysis, suggesting that the increase in procollagen mRNA with TGFβ1 treatment was due to a post‐transcriptional process(es). However, both transcriptional and post‐transcriptional components were observed in the regulation of fibronectin gene expression by TGFβ1. In addition, TGFβ1 was found to positively regulate its own expression, as treatment of the cells with TGFβ1 enhanced endogenous TGFβ1 expression and prevented the small decrease in TGFβ1 mRNA levels which occurred early during the differentiation program. Thus, our data demonstrate that down‐regulation of type I procollagen, fibronectin, and TGFβ1 gene expression was prevented during TGFβ inhibition of 3T3‐L1 differentiation. Taken together, these data suggest that TGFβ may inhibit differentiation of 3T3‐L1 cells by maintaining the fibroblast‐like extracellular matrix, thus preventing the changes in cell shape that accompany differentiation.
Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up‐regulation of oncogenes in S‐49 cyc− cells
Authors: Hughes‐Fulford Millie
Published: May 2005

Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c‐AMP in prostaglandin mediated cell cycle arrest, we use the‐49 lymphoma variant (cyc−) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A1 (dmPGA1) inhibits DNA synthesis and cell growth in cyc− cells. DNA synthesis is inhibited 42% by dmPGA1 (50 μM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the α,β unsaturated ketone ring. Dimethyl PGA1 is most effective in inhibiting DNA synthesis in cyc− cells, with prostaglandins PGE1 and PGB1 being less potent inhibitors of DNA synthesis. DmPGE2 caused a significant stimulation of DNA synthesis. S‐49 cyc‐ variant cells exposed to (30–50 μm) dmPGA1, arrested in the G1 phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S‐49 cyc− cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G1, S, G2, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S‐phase DNA synthesis from the G1 cell cycle block. The S‐49 cyc− cells are known to have a G1/S boundary through M phase transition time of 14.8 h, making the location of the prostaglandin cell cycle arrest at or very near the G1/S interface. The oncogenes, c‐fos and c‐myc which are normally expressed during G1 in proliferating cells have a 2–3 fold enhanced expression in prostaglandin G1 arrested cells. These data using the S‐49 variants demonstrate that dmPGA1 inhibits DNA synthesis and arrests the cell cycle independent of cAMP‐mediated effects. The prostaglandin arrested cells maintain the gene expression of a G1 synchronous cell which suggests a unique molecular mechanism for prostaglandin action in arresting cell growth. These properties indicate that this compound may be an effective tool to study molecular mechanisms of regulation of the cell cycle.
Isolation of cDNA clones from an osteosarcoma‐ROS17/2.8 library by differential hybridization
Authors: Waye Mary M. Y. , Li Vincent K. C.
Published: May 2005

We have used differential hybridization to isolate and characterize two novel cDNAs expressed in chondrocytes and some osteoblastic cells. A rat osteosarcoma ROS17/2.8 cDNA library was screened and cDNA clones hybridizing strongly to radiolabeled porcine calvaria cDNA but weakly to a control radiolabeled cDNA were isolated. Two clones were obtained—p.6.1 and p.10.15. A radiolabeled probe of p10.15 was shown to hybridize specifically to a 2.3 Kb message RNA from a chondrogenic clonal cell population from rat calvaria‐RCJ 3.1C5.18, and the mRNA was downregulated by 1,25 (OH)2D3, which inhibits chondrogenesis in these cells. The other clone, p6.1, was found to hybridize to a 0.95 Kb message that is expressed in rat liver, kidney, lung, muscle, and brain, but not expressed in spleen and expressed only in low levels in thymus.
Cell attachment peptide of C‐reactive protein: critical amino acids and minimum length
Authors: Mullenix Michael C. , Kaumaya Pravin T. P. , Mortensen Richard F.
Published: May 2005

Human C‐reactive protein (CRP) is an acute phase blood component that accumulates at sites of tissue damage and necrosis and is degraded by neutrophils to biologically active peptides. A dodecapeptide composed of amino acids 27–38 of CRP mediates cell attachment in vitro. This peptide was designated the cell‐binding peptide (CB‐Pep) of CRP. Characterization of the interaction between fibroblasts and modified synthetic peptides with sequential deletions from either the N‐terminus or C‐terminus revealed that the minimal sequence for cell attachment or inhibition of cell attachment to the CB‐Pep was Phe‐Thr‐Val‐Cys‐Leu, which corresponds to residues 33–37 within each of the five 206 amino acid subunits of CRP. The pentapeptide by itself mediated cell attachment. Substitutions for each residue within the CB‐Pep indicated that the critical residues for activity were Phe‐33 and Thr‐34. This cell‐binding pentapeptide represents a recognition motif for cell adhesion not found in other proteins.
Histone gene transcription: A model for responsiveness to an integrated series of regulatory signals mediating cell cycle control and proliferation/differentiation interrelationships
Authors: Stein Gary S. , Stein Janet L. , van Wijnen André J. , Lian Jane B.
Published: May 2005

Histone gene expression is restricted to the S‐phase of the cell cycle. Control is at multiple levels and is mediated by the integration of regulatory signals in response to cell cycle progression and the onset of differentiation. The H4 gene promoter is organized into a series of independent and overlapping regulatory elements which exhibit selective, phosphorylation‐dependent interactions with multiple transactivation factors. The three‐dimensional organization of the promoter and, in particular, its chromatin structure, nucleosome organization, and interactions with the nuclear matrix may contribute to interrelationships of activities at multiple promoter elements. Molecular mechanisms are discussed that may participate in the coordinate expression of S‐phase‐specific core and H1 histone genes, together with other genes functionally coupled with DNA replication.