bluuebunny/crossref_metadata_2025 huggingface dataset:
EXPRESSION AND PURIFICATION OF BIOLOGICALLY ACTIVE RECOMBINANT QUAIL STEM CELL FACTOR IN E. COLI
Authors: D'Costa Susan , Kulik Michael J. , Petitte James N.
Published: September 2002

Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. Previous studies have demonstrated that avian SCF is a requirement for the proliferation and survival of various cell types in vivo and in vitro. In the current study, recombinant quail stem cell factor was produced inEscherichia coli using a prokaryotic expression system. SCF was expressed as a fusion protein with a histidine hexamer tag at the N‐terminal end of the protein. Following expression, the protein was purified by affinity chromatography on the Ni‐NTA column. The uninduced and induced protein lysates and the purified protein were separated by SDS‐PAGE and transferred onto nitrocellulose membrane. Western blot analysis with the monoclonal antibody to the histidine tag identified SCF in the induced cell lysates and the purified sample. The recombinant SCF was approximately 22–23kD in size. This protein was generated devoid of the signal peptide, the transmembrane domain, and the intracellular domain and, hence, resembles the soluble form of SCF. Biological activity was assayed using the in vitro survival of E12 chicken dorsal root ganglion‐derived sensory neurons. The addition of recombinant quail SCF improved neuronal survival. Survival (20.6%) was the highest at the 50ng/ml concentration of SCF. The availability of quail SCF will be a valuable tool to further resolve the function of stem cell factor in birds.
GALECTIN‐3 EXPRESSION IN DIFFERENTIATING HUMAN MYELOID CELLS
Authors: Le Marer Nadia
Published: September 2002

Galectin‐3 is an endogenous mammalian carbohydrate‐binding protein with affinity for ABH group carbohydrate epitopes and polylactosamine glycans present on cell surface and extracellular matrix glycoproteins. It has been shown to play a role in cell differentiation, morphogenesis, adhesion and cell proliferation regulation. Progenitor cell proliferation in bone marrow depends on stem cell factors including those modulating their adhesion to the bone marrow stroma. The present study shows that the 32kD galectin‐3 is developmentally expressed in human myeloid cells and is strongly upregulated on the cell surface of late mature myeloid cells. Despite the fact that the expression of the galectin‐3 is very low in CD34+ early myeloid cell, a 70kD protein is found by Western blotting using antibodies specific to galectin‐3, exclusively in those cells. Finally, exogenous human recombinant galectin‐3 binds strongly to CD34+ early myeloid cells and emphasizes granulocyte‐colony stimulating factor (G‐CSF) driven proliferation in vitro.
SPEEDING UP KINESIN‐DRIVEN MICROTUBULE GLIDING IN VITRO BY VARIATION OF COFACTOR COMPOSITION AND PHYSICOCHEMICAL PARAMETERS*
Authors: Böhm K. J. , Stracke R. , Unger E.
Published: September 2002

So far, there has been a discrepancy between the velocities of kinesin‐dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg2+, and kinesin concentrations, pH and ionic strength, we tried to find conditions that favour microtubule gliding across kinesin‐covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg2+/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temperature elevation or lowering the number of kinesin molecules bound to the glass surface. The highest mean gliding velocity (1.8μm/s±0.09μm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favourable factors (2.5m m ATP, 12.5m m Mg2+, 37°C, 450 kinesin molecules/μm2).
ROLE OF INTERLEUKIN‐6 IN THE PATHOGENESIS OF MULTIPLE MYELOMA
Authors: Gadó Klara , Domján Gyula , Hegyesi Hargita , Falus András
Published: September 2002

Multiple myeloma (MM) is a currently incurable disease caused by the proliferation of malignant plasma cells. Although the pathogenesis of the disease still remains unclear, recent research in the biology of MM has produced new insights into the factors that control the growth and survival of myeloma cells. Among the growth factors, interleukin‐6 (IL‐6) has an essential role. Evidence suggests that IL‐6 is not only a growth factor, but also a survival factor in MM, inhibiting apoptosis in myeloma cells. IL‐6 interacts with several factors which are involved in the pathogenesis of MM, such as adhesion molecules, tumour suppressor genes and oncogenes. Considering the essential role of IL‐6, it could serve as a target for new therapeutic interventions. Neutralizing the effect of IL‐6 may result in a regression of tumour progression.
SATELLITE CELL REGULATION FOLLOWING MYOTRAUMA CAUSED BY RESISTANCE EXERCISE
Authors: Vierck Janet , O'Reilly Becky , Hossner Kim , Antonio Jose , Byrne Katherine , Bucci Luke , Dodson Michael
Published: September 2002

It is generally accepted that the primary mechanisms governing skeletal muscle hypertrophy are satellite cell activation, proliferation, and differentiation. Specific growth factors and hormones modulate satellite cell activity during normal muscle growth, but as a consequence of resistance exercise additional regulators may stimulate satellite cells to contribute to gains in myofiber size and number. Present knowledge of the regulation of the cellular, biochemical and molecular events accompanying skeletal muscle hypertrophy after resistance exercise is incomplete. We propose that resistance exercise may induce satellite cells to become responsive to cytokines from the immune system and to circulating hormones and growth factors. The purpose of this paper is to review the role of satellite cells and growth factors in skeletal muscle hypertrophy that follows resistance exercise.
SPECTRIN‐LIKE PROTEINS IN PLANT NUCLEI
Authors: De Ruijter Norbert C. A. , Ketelaar Tijs , Blumenthal Sonal S. D. , Emons Anne Mie C. , Schel Jan H. N.
Published: September 2002

We analysed the presence and localization of spectrin‐like proteins in nuclei of various plant tissues, using several anti‐erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross‐reactive pair of bands at 220–240kDa, typical for human erythrocyte spectrin, and a prominent 60kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin‐like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti‐spectrin antibodies used. In some nuclei tracks of spectrin‐like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin‐like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin‐like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.
STRUCTURAL ASSOCIATION OF BAX WITH NUCLEAR MATRIX AND CYTOMATRIX REVEALED BY EMBEDMENT‐FREE IMMUNOGOLD ELECTRON MICROSCOPY
Authors: Gajkowska Barbara , Motyl Tomasz , Olszewska‐Badarczuk Hanna , Gniadecki Robert , Koronkiewicz Mirosława
Published: September 2002

Bax is a cellular protein functioning as a promoter of apoptosis. It is ultrastructurally associated with mitochondrial membranes, where it participates in permeability transition pore formation. By employing embedment‐free electron microscopy (EFEM), we present evidence that Bax is also associated with the nuclear matrix and cytomatrix of cultured human tumour cells (COLO 205, PA‐1, U‐373 MG). Extracted cellular scaffolds were probed with anti‐Bax antibody using the immunogold electron microscopy technique. Bax immunoreactivity was found on 10–15nm intermediate filaments of karyo‐ and cytoskeleton, stretched between the nucleus, nuclear lamina and cell periphery. Bax immunoreactivity was preferentially localized to certain areas of filaments (spot‐like). The target molecules for Bax binding in the cellular matrix and their physiological significance remain to be established.
CYTOKINES IN DISEASES OF THE ENDOCRINE SYSTEM
Authors: Igaz Peter , Falus András , Gláz Edit , Rácz Károly
Published: September 2002

Cytokines, the polypeptide mediators of the immune system, were shown to exert numerous actions on endocrine functions. Bidirectional links based on the sharing of mediators and receptors between the immune and neuroendocrine systems lead to the concept of the immune—neuroendocrine system that seems to constitute an important and sophisticated regulatory system in the homeostasis. Several cytokines were found to be involved in the pathogenesis of diseases of the endocrine system. In this brief review, we attempt to present a general outline of the local actions of cytokines on cells of endocrine organs with an emphasis on disease etiology (pituitary tumours and autoimmune endocrine diseases in particular).
CATALASE ACTIVITY IN HUMAN ERYTHROCYTES: EFFECT OF PHENOXYHERBICIDES AND THEIR METABOLITES
Authors: Bukowska Bozena , Chajdys Anna , Duda Wirgiliusz , Duchnowicz Piotr
Published: September 2002

The effects of exposure to different concentrations of phenoxyherbicides and their metabolites were studied in human erythrocytes, with particular attention to catalase (CAT—EC. 1.11.1.6— hydrogen peroxide: hydrogen peroxide oxidoreductase). 4‐chloro‐2‐methylphenoxyacetic acid (MCPA), 2,4‐dimethylphenol (2,4‐DMP) and 2,4‐dichlorophenoxyacetic acid (2,4‐D) did not affect CAT activity, but 2,4‐dichlorophenol (2,4‐DCP) and 2,4,5‐trichlorophenol (2,4,5‐TCP) decrease its activity, the latter being the more inhibitory.
CHAOTIC DYNAMICS AND FRACTAL SPACE IN BIOCHEMISTRY: SIMPLICITY UNDERLIES COMPLEXITY
Authors: Aon Miguel A. , Cortassa Sonia , Lloyd David
Published: September 2002

In this work we attempt to analyze the coupling between the dynamics of biochemical reactions (especially chaotic dynamics), and the geometry of cytoarchitecture (especially fractal ultrastructure), because of its importance and consequences for the ultradian dynamic behaviour of cells. Fractal geometry in intracellular macromolecular assemblies suggests that chaotic dynamics occur during their organization. Non‐linear interactions in and between spatial and temporal domains and over wide ranges of scales underlie the emergent properties of complex biological systems.
EFFECT OF FGF‐1 AND FGF‐2 ON VEGF BINDING TO HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS
Authors: Chen Jun‐Hui , Wang Xin‐Chang , Kan Mikio , Sato J. Denry
Published: September 2002

When FGF‐1 or FGF‐2 and VEGF were added together, the mitogenic effect of FGF‐1 or FGF‐2 and VEGF on HUVEC was additive. However, when HUVECs were preincubated for 2 days with 10ng/ml FGF‐1 in the absence of VEGF, the Scatchard plot of [125I]VEGF binding sites was shifted to the right: both affinity classes of VEGF binding sites were equally affected, such that the total number of sites increased twofold. It is suggested that this type of interaction may be related to tumor angiogenesis and wound repair.
POSSIBLE ROLE OF FAS ANTIGEN (CD 95) IN HUMAN AMNIOTIC EPITHELIAL CELL DEATH: AN IN VITRO STUDY
Authors: Surendran Sankar
Published: September 2002

Treatment of human amniotic epithelial (HAE) cells with anti‐Fas monoclonal antibody (CH 11) at 100ng/ml or 1μg/ml for 12 or 24h increased necrotic cell death. Apoptotic cell death induced by this antibody was significantly increased, although far fewer cells underwent apoptosis, as determined by the TUNEL method. This study suggests that Fas antigen is an important mediator in HAE cell death.
PERSISTENT FUSION PORES BUT TRANSIENT FUSION IN ALVEOLAR TYPE II CELLS
Authors: Dietl Paul , Haller Thomas
Published: September 2002

The release of vesicle contents following exocytotic fusion is limited by various factors including the size of the fusion pore. Fusion pores are channel‐like, narrow structures after formation and proceed through semi‐stable states (‘fusion pore flickering’), unless they fully expand (full fusion) or close again (transient fusion). Partial release of vesicle contents may occur during transient fusion, which was described to last between milliseconds and seconds, depending on the size of the vesicle. We studied fusion pores in a slow‐secreting lung epithelial cell (type II cell) using fluorescence staining of vesicle contents (surfactant) and fluorescence recovery after photobleaching (FRAP). Surfactant is a lipidic material, which is secreted into the alveolar lumen to reduce the surface tension in the lung. We found release of surfactant to be a slow process, which can last for hours. Accordingly, fusion pores in these cells are stable structures, which appear to be a barrier for release. FRAP measurements suggest that transient fusions occasionally take place in these long‐lasting fusion pores, resulting in partial release of surfactant into the extracellular space. These data suggest that postfusion mechanisms may regulate the amount of secreted surfactant.
DEPLOYMENT OF MEMBRANE FUSION PROTEIN DOMAINS DURING FUSION
Authors: Bentz Joe , Mittal Aditya
Published: September 2002

It is clear that both viral and intracellular membrane fusion proteins contain a minimal set of domains which must be deployed at the appropriate time during the fusion process. An account of these domains and their functions is given here for the four best‐described fusion systems: influenza HA, sendai virus F1, HIV gp120/41 and the neuronal SNARE core composed of synaptobrevin (syn), syntaxin (stx) and the N‐ and C‐termini of SNAP25 (sn25), together with the Ca2+binding protein synaptotagmin (syt). Membrane fusion begins with the binding of the virion or vesicle to the target membrane via receptors. The committed step in influenza HA‐ mediated fusion begins with an aggregate of HAs (at least eight) with some of their HA2 N‐termini, a.k.a. fusion peptides, embedded into the viral bilayer (Bentz, 2000 a). The hypothesis presented in Bentz (2000 b) is that the conformational change of HA to the extended coiled coil extracts the fusion peptides from the viral bilayer. When this extraction occurs from the center of the site of restricted lipid flow, it exposes acyl chains and parts of the HA transmembrane domains to the aqueous media, i.e. a hydrophobic defect is formed. This is the ‘transition state’ of the committed step of fusion. It is stabilized by a ‘dam’ of HAs, which are inhibited from diffusing away by the rest of the HAs in the aggregate and because that would initially expose more acyl chains to water. Recruitment of lipids from the apposed target membrane can heal this hydrophobic defect, initiating lipid mixing and fusion. The HA transmembrane domains are required to be part of the hydrophobic defect, because the HA aggregate must be closely packed enough to restrict lipid flow. This hypothesis provides a simple and direct coupling between the energy released by the formation of the coiled coil to the energy needed to create and stabilize the high energy intermediates of fusion. Several of these essential domains have been described for the viral fusion proteins SV5 F1 and HIV gp120/41, and for the intracellular SNARE fusion system. By comparing these domains, we have constructed a minimal set which appears to be adequate to explain how the conformational changes can produce a successful fusion event, i.e. communication of aqueous compartments.
PROTEIN TYROSINE KINASE‐DEPENDENT REGULATION OF ADENYLATE CYCLASE AND PHOSPHATIDYLINOSITOL 3‐KINASE ACTIVATES THE EXPRESSION OF GLIAL FIBRILLARY ACIDIC PROTEIN UPON INDUCTION OF DIFFERENTIATION IN RAT C6 GLIOMA
Authors: Roymans D. , Grobben B. , Claes P. , Slegers H.
Published: September 2002

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP‐mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or β‐adrenergic receptor‐mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3‐kinase (PI 3‐K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3‐K activity in C6 glioma cells.
CLONING AND EXPRESSION OF MUNC 30: A MEMBER OF THE PAIRED ‐LIKE HOMEODOMAIN GENE FAMILY
Authors: Nicholson L. F. B. , Ma L. , Goulding M.
Published: September 2002

The temporal and spatial expression of transcription factors is of critical importance in the organization and specification of cellular phenotypes in the anterior structures of the head and in particular the CNS. In order to identify further genes which might play a role in such patterning we have cloned the Munc 30 gene, a new isoform of the paired ‐like homeodomain gene Ptx2. Using RT—PCR, Munc 30 expression was detected in embryonic head and brain and in a wide panel of adult mouse tissues including brain, spinal cord, eye and tongue. In situ hybridization showed the expression domain of Munc 30 to be localized to a wide variety of developing organs and primordial tissues of the embryo with extremely high levels of expression in Rathke's pouch, tooth primordia and the hypothalamus. In situ RT—PCR was used to localize gene expression to cells of the cortex, striatum and thalamus of adult mouse forebrain. Together, these expression patterns suggest that this gene may not only play a critical role in patterning of anterior structures of the head during development but may also be responsible for the maintenance and/or modulation of cell identity in adult.
LOW DOSES OF BIOLOGICALLY ACTIVE SUBSTANCES: EFFECTS, POSSIBLE MECHANISMS, AND FEATURES
Authors: Gurevich Konstantin G.
Published: September 2002

Some substances can produce a response in biological systems at extraordinarily low concentrations. Often called the ‘low’ or ‘small’ dose effect, the meaning or definition of what is meant by ‘low’ in this context is discussed. For the purposes of this article the expression is taken to be a concentration substantially below the equilibrium dissociation constant of the effector‐target complex, but extremely low concentrations (<10−19M) are excluded from consideration. The main features of very low or ‘small’ dose action are described. Possible mechanisms of the effects of very low doses are suggested. The main thrust of the paper concerns the explanation of bimodal and polymodal dose‐effect curves. A general formal kinetic model for such curves and its application will be discussed.
ALTERED MATRIX METALLOPROTEINASE EXPRESSION ASSOCIATED WITH ONCOGENE‐MEDIATED CELLULAR TRANSFORMATION AND METASTASIS FORMATION
Authors: Baruch Robert R. , Melinscak Hrovje , Lo James , Liu Yang , Yeung Oliver , Hurta Robert A. R.
Published: September 2002

Matrix metalloproteinase expression was examined in a series of mammalian cell lines of varying degrees of malignant progression. The expression of MMP‐2 and MMP‐9 was found to correlate with ras‐mediated cellular transformation and as a function of malignant potential. Altered MMP‐2 and MMP‐9 expression was found to correlate also in other oncogene transformed cell lines and the level of expression of both MMP‐2 and MMP‐9 correlated with metastatic potential. Increased expression of both MMP‐2 and MMP‐9 was also found in cells which constitutively over‐express MAP kinase kinase suggesting that one of the consequences of the persistent activation of the MAP kinase pathway is elevated expression of MMP‐2 and MMP‐9. Additionally, this study demonstrated a correlation between the expression of MMP‐3 (stromelysin‐1) and the level of ras expressed in cells and with the cells' ability to form tumors and with malignant potential. The existence of a novel 80kDa caseinase activity which correlates with ras expression and the ability of the cell to form tumors was also demonstrated. The growth status of transformed cells was also found to be important in determining the expression of MMP‐2 mRNA but not MMP‐9 mRNA expression, and this expression was cell‐type specific. This study also demonstrates that oncogenes can interact to influence and to determine the nature of the matrix metalloproteinases expressed and that this interaction results in a tumorigenic phenotype and, most importantly, contributes to the metastatic phenotype. Alterations in the expression and the regulation of MMPs, particularly MMP‐2 and MMP‐9, constitute an integral part of the altered growth regulatory program found within transformed cells and in particular, in transformed cells capable of malignant progression.
TYROSINE PHOSPHORYLATION TRANSLOCATES β‐CATENIN FROM CELL—CELL INTERFACE TO THE CYTOPLASM, BUT DOES NOT SIGNIFICANTLY ENHANCE THE LEF‐1‐DEPENDENT TRANSACTIVATING FUNCTION
Authors: Kim Kwonseop , Lee Kwang‐Youl
Published: September 2002

β‐catenin plays an essential role in cells, not only as a cadherin‐associated complex, but also as a signaling molecule in the nucleus. Tyrosine phosphorylation of β‐catenin has been shown to correlate with tumorigenesis, cell migration, and developmental processes. However, its exact effects on downstream targets in the nucleus are not yet clear. In this study, we used HCT‐15 colon carcinoma and NIH 3T3 fibroblasts as models to investigate the effects of a phosphotyrosine phosphatase (PTPase) inhibitor on the localization of β‐catenin, the binding affinity to LEF‐1 (Lymphoid Enhancer Factor), and on LEF‐1‐dependent transactivation function. Treatment with a PTPase inhibitor, pervanadate, increased the tyrosine phosphorylation of β‐catenin in a time‐dependent manner and led to its relocation from cell—cell interfaces to the cytoplasm. This phosphorylation/dephosphorylation of β‐catenin does not require its presence at cell—cell interfaces. However, tyrosine phosphorylation of β‐catenin does not change its binding affinity to LEF‐1 nor enhance cyclin D1 transactivation, a nuclear target of β‐catenin/LEF‐1. This result suggests that tyrosine phosphorylation of β‐catenin has effects on the binding to cadherins in the cytoplasm but not on its LEF‐1‐dependent transactivating function in the nucleus.
TUMOUR NECROSIS FACTOR‐α PROVOKES UPREGULATION OF α2β1 AND α5β1 INTEGRINS, AND CELL MIGRATION IN OST OSTEOSARCOMA CELLS
Authors: Kawashima Atsuhiro , Kawahara Ei , Tokuda Ryouko , Nakanishi Isao
Published: September 2002

OST cells enhance the induction of matrix metalloproteinase (MMP)‐9 by tumour necrosis factor (TNF)‐α and the corresponding metastasis to lungs in vivo (Kawashima et al., 1994). We focused on the adhesive and migratory properties of OST cells, and investigated the expression of integrins in OST cells stimulated by TNFαin vitro. OST cells potentiated not only adhesion to the extracellular matrix (ECM) but also the migration on ECM. On competitive reverse transcription‐polymerase chain reaction (RT‐PCR) analyses, the amounts of α2 (4.9‐fold), α5 (1.2‐fold) and αv (4.9‐fold) were upregulated by TNFα at the transcriptional level. Alpha‐5 showed a slight increase by flow cytometry; however, α2 and αv integrins remained unchanged at the protein level. Immunofluorescence study disclosed integrins of α2β1 and α5β1 were much clustered at cell processes by TNFα stimulation, probably related to increased cell adhesion and migration. Therefore, the upregulation of α2β1 and α5β1 integrins seems to contribute to tumour invasion and metastatic potential.
CHARACTERIZATION OF MYOGENIC CELL MEMBRANE: SPONTANEOUS FORMATION OF HETEROKARYOTIC MYOTUBES BETWEEN TWO DIFFERENT KINDS OF MYOBLASTS
Authors: Hirayama Etsuko , Nakanishi Masami , Kim Jeman
Published: September 2002

In a previous study, it has been shown that presumptive mouse C2 myoblast cells are strongly resistant to HVJ (hemaglutinating virus of Japan, Sendai virus)‐mediated cell fusion, but do become capable of fusion upon differentiation. Quail myoblasts transformed with a temperature‐sensitive mutant of Rous sarcoma virus (QM‐RSV cells) also become more sensitive to HVJ‐mediated cell fusion during differentiation. Investigations were undertaken to see whether heterokaryotic myotubes were formed spontaneously by co‐culture of two different kinds of myogenic cells, QM‐RSV cells and C2 cells. When both cells were committed to myotube formation, they spontaneously fused without HVJ on co‐culture. On the other hand, when both or one of the cells were in the presumptive state, heterokaryons were not formed by co‐culturing. Furthermore, committed QM‐RSV cells did not fuse with non‐myogenic cells. These results indicate that the membranes of myogenic cells change to become capable of fusion for myotube formation during differentiation.
UTILIZATION OF AMINO ACIDS IN GROWING KIDNEY PROXIMAL TUBULE CELL CULTURES
Authors: Ali Abdi Rashid , Evans Peter J.
Published: September 2002

The growth of rat kidney proximal tubule cells was monitored continuously by the cellular incorporation of [methyl‐14C] thymidine using scintillating microplates. The radioisotope had no effect on cell proliferation over a 5 day period, neither was it extensively converted to thymine. Leibovitz L‐15 medium supplemented with bicarbonate proved a good growth medium and its high levels of carbohydrates and amino acids facilitated the appearance of intermediates in the cells' metabolism of additional radioactive amino acids. Kidney proximal tubule cells had a greater potential to process amino acids than BHK‐21 cells. The utilization of amino acids by proximal tubule cells differed from that of other organs. The amino acids could be classified into three classes. Members of the first type were only used for protein synthesis (arginine, lysine, histidine and tyrosine). The second class of amino acids yielded only one or two metabolites (leucine and isoleucine), while the last type gave more than two metabolites (alanine, aspartate, glycine, methionine, proline and valine).
NOT ALL MUSCLES MEET THE SAME FATE WHEN THEY DIE
Authors: Jones Margaret E. E. , Schwartz Lawrence M.
Published: September 2002

Two general patterns of cell death are usually described in animals: necrosis and apoptosis. The former is a passive process that displays cellular swelling and lysis, while the latter involves cellular shrinkage and gene‐mediated, ATP‐dependent processes. Independent of the proximal cause of cell death, cell corpses are almost always removed by phagocytic cells. This is far from universal for all cells however, since phagocytic cells have not been noted during the programmed death of some skeletal muscles in insects. To further explore this, we used a variety of anatomical methods to examine the death of the intersegmental muscles (ISMs) of the moth Manduca sexta. The ISMs are giant cells that die during the 30h following adult emergence. At no stage examined were hemocytes or other cells associated with the sarcolemma. The failure to detect macrophages was not due to technical limitations since immunohistochemical and functional studies demonstrate their presence in the hemolymph. The absence of phagocytosis to remove ISM corpses suggests that all of the biochemical machinery required for cellular destruction is resident within the ISMs themselves. This is consistent with analysis suggesting that Manduca does not possess sufficient numbers of macrophages to consume the ISMs. Given that insects do not have adaptive immunity, the ability to use a completely cell autonomous process may be a developmental option that cannot be exploited in vertebrates.
MYCOBACTERIAL GROWTH IN HUMAN MACROPHAGES: VARIATION ACCORDING TO DONOR, INOCULUM AND BACTERIAL STRAIN
Authors: Hoal‐van Helden E. G. , Hon D. , Lewis L.‐A. , Beyers N. , van Helden P. D.
Published: September 2002

The microbicidal capacity of the macrophage is frequently evaded by mycobacteria, leading to tuberculosis (TB). We investigated a number of parameters affecting the rate of growth of mycobacteria in human monocyte‐derived macrophages (MDM). The results show a great deal of variation in the growth of both Mycobacterium bovis BCG and M. tuberculosis H37Rv, using a large number of human macrophage donors, (132 and 40, respectively), but no correlation was seen with the TB status of the MDM donor. Clumping of the mycobacteria resulted in more vigorous growth in MDM, suggesting that inoculum size could affect disease progression. The growth rates of 17 clinical isolates of M. tuberculosis were measured in macrophages derived from three donors and no consistent or marked differences between isolates were observed over the 5‐day period of growth measurement. However, all 17 clinical strains grew consistently faster than H37Rv in the same experiments.
DIVERSITY OF IN VITRO CYTOKINE RESPONSES BY HUMAN MACROPHAGES TO INFECTION BY MYCOBACTERIUM TUBERCULOSIS STRAINS
Authors: Hoal‐van Helden E. G. , Stanton L.‐A. , Warren R. , Richardson M. , van Helden P. D.
Published: September 2002

Macrophages are an important component in the first line of defence of the innate immune system. They are capable of producing cytokines in response to bacterial challenge, as well as in response to cytokine stimuli from other cells in the immune system. The microbicidal response of human monocyte‐derived macrophages in vitro, induced by exogenously added cytokines, is highly variable. We found that tumour necrosis factor‐α (TNF‐α) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) could have either stimulatory or inhibitory effects on intracellular BCG killing, depending on the macrophage donor. Macrophages infected in vitro by various clinical isolates of Mycobacterium tuberculosis or the laboratory strain H37Rv, produced varying levels of both TNF‐α and IFN‐γ. Certain M. tuberculosis strains tended to be associated with high cytokine production in each of three independent experiments, indicating that strains may differ in the host response elicited to infection.
BRIEF OCCLUSION OF THE MAIN PANCREATIC DUCT RAPIDLY INITIATES SIGNALS WHICH LEAD TO INCREASED DUCT CELL PROLIFERATION IN THE RAT
Authors: Ferris W. F. , Woodroof C. W. , Louw J. , Wolfe‐Coote S. A.
Published: September 2002

In the course of investigating the signals associated with pancreas regeneration, we have developed a method to initiate pancreatic duct cell proliferation by brief occlusion of the main pancreatic duct. The resulting duct cell proliferation, induced by temporary partial main duct occlusion, was compared to that induced by firmly tying a cellophane strip around the head of the pancreas for longer periods of time. Both methods stimulated a biphasic increase in duct cell proliferation, with proliferation maxima at 3 and 14 days post operation. The short duration of temporary main duct occlusion (60s) that was needed to stimulate duct cell proliferation, and the similar duct cell proliferation profiles that were observed after both the temporary and the longer term main duct occlusion, led us to conclude that the signals which initiate proliferation occur rapidly at the beginning of each procedure.
EFFECTS OF TH‐2 TYPE CYTOKINES ON HUMAN AIRWAY EPITHELIAL CELLS: INTERLEUKINS‐4, −5, AND −13
Authors: Relova A. J. , Kampf C. , Roomans G. M.
Published: September 2002

Proliferation and death of airway epithelial cells may be of importance in the pathogenesis of asthma. T‐helper (Th)‐2 response interleukins (IL)‐4, −5, and −13 have the ability to induce proliferation of airway epithelial cells.
PURIFICATION AND IDENTIFICATION OF A HUMAN DERMAL EXTRACT COMPONENT INHIBITORY TO FIBROBLAST PROLIFERATION
Authors: Stewart Joanne E. , Wheatley D. N. , Holmes J. D. , Muir I. F. K.
Published: September 2002

It was previously shown that a citric acid buffer extract of human dermis (extract D) inhibited growth of human diploid fibroblasts in monolayer culture (Muir et al., 1997). Further fractionation has shown that the active principle is probably a proteoglycan, and that retention of its inhibitory activity is dependent on the use protease inhibitors throughout the extraction procedure. Elution of extract D from a DEAE‐cellulose column produced four major peaks, each of which was subjected to SDS‐PAGE as well as being tested for inhibitory activity on the growth of fibroblasts in culture. Peaks III and IV had no inhibitory effect, but peak I contained highly active material. Gels of this peak showed prominent bands of 120kDa (corresponding to dermatan sulphate proteoglycan II, DS‐PG II) and at 45kDa (corresponding to the core protein). The latter band became more prominent when extract D which had been treated with chrondroitinase ABC was electrophoresed. Their identities were verified by Western blotting. Peak II also contained some slower‐acting inhibitory material which has as yet to be identified, but contains little or no protein corresponding to the decorin core‐protein. The data indicate that the intact decorin molecule, DS‐PG II, is the main inhibitory principle in human skin.
INHIBITION OF THE CONTRACTION OF COLLAGEN GELS BY EXTRACTS FROM HUMAN DERMIS
Authors: El‐Sheemy M. A. , Muir I. F. K. , Wheatley D. N. , Eremin O.
Published: September 2002

Extracts from the human dermis were prepared and evaluated with respect to their ability to influence fibroblasts to contract collagen gels in vitro. The extract which had the most inhibitory effect on fibroblasts to cause contraction of collagen gels was extract D. It also inhibited fibroblast growth. Inhibition of contraction was not simply related to fibroblast cell numbers and the data suggests a specific effect upon the ability of fibroblasts to cause contraction. Other extracts were without significant effect.
CLONING, SEQUENCING AND EXPRESSION OF A cDNA ENCODING THE MOUSE L35a RIBOSOMAL PROTEIN DURING DIFFERENTIATION OF MURINE ERYTHROLEUKEMIA (MEL) CELLS
Authors: Pappas I. S. , Vizirianakis I. S. , Tsiftsoglou A. S.
Published: September 2002

In a previous study we reported that ribosomal protein S5 gene is suppressed in differentiating and not in proliferating or apoptotic murine erythroleukaemia (MEL) cells (Vizirianakis et al., 1999). We wish to report here the isolation, characterisation and expression of the full length cDNA for another ribosomal protein, the L35a (rpL35a), in MEL cells. This cDNA shares significant structural homology in both DNA and protein levels to genes encoding the rat and human L35a ribosomal proteins. Northern blot hybridisation analysis has shown that the steady‐state level of rpL35a mRNA is progressively reduced during differentiation of MEL cells along the erythrocytic maturation pathway induced by DMSO or UDP‐4, two structurally unrelated inducers of differentiation. However, in cells where differentiation was inhibited by N6‐methyladenosine, the level of rpL35a RNA transcripts was not affected. In addition, rpL35a gene expression was not altered in apoptotic MEL cells. Furthermore, the suppression of L35a gene was not correlated to any change in DNA methylation at CCGG sites located at the rpL35a gene locus in undifferentiated and differentiated MEL cells, as we observed for the rpS5 gene. Overall, these data suggest that the expression of ribosomal genes, the L35a of 60S ribosomal subunit and the S5 of 40S ribosomal subunit, are regulated by a common mechanism in differentiating MEL cells, leading to the observed decrease in ribosomal function.
PHOSPHORYLATION OF FILAMIN (ABP‐280) REGULATES THE BINDING TO THE LIPID MEMBRANE, INTEGRIN, AND ACTIN
Authors: Goldmann Wolfgang H.
Published: September 2002

Actin‐binding protein (ABP‐280; filamin) is a phosphoprotein present in the periphery of the cytoplasm, where it can cross‐link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, the hypothesis that it serves as a substrate for p56lck, a lymphocyte‐specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins is considered. The results suggest conformationally‐induced regulation of filamin (ABP‐280).
X‐RAY MICROANALYSIS OF ETOPOSIDE‐INDUCED APOPTOSIS IN THE PC‐3 PROSTATIC CANCER CELL LINE
Authors: Salido M. , Vilches J. , Lopez A. , Roomans G. M.
Published: September 2002

Apoptosis comprises a critical intracellular defense mechanism against tumourigenic growth. We have been interested in the relationship between morphological changes and intracellular concentration of several cations after etoposide‐induced apoptosis in androgen‐independent prostate cancer cells. SEM and X‐ray microanalysis were performed on freeze‐dried PC3 cells after etoposide treatment, and correlated with the morphological features observed after examination by light and fluorescence microscopy. Cell viability assays were also performed. A significant decrease in intracellular Cl−and K+and a progressive increase in Mg2+and Na+were observed, with parallel changes in cellular volume as cells passed through three morphological stages of apoptosis. The use of EPXRMA made it possible to evaluate alterations in element composition in prostate cancer cell apoptosis and may be a helpful tool for further studies on apoptosis in prostate cancer.
THYROID‐STIMULATING HORMONE AND GROWTH HORMONE RELEASE ALTERATIONS INDUCED BY MOSQUITO LARVAE PROTEINS ON PITUITARY CELLS
Authors: Bolognani Federico , Goya Rodolfo Gustavo , Ronderos Jorge Rafael
Published: September 2002

Mosquito larvae crude extract has shown to modulate cell proliferation of different mouse epithelial as well as human mononuclear cell populations in vivo and in vitro. A soluble fraction of the extract, with a molecular weight ranging from 12 to 80kD, also showed an inhibitory effect on the proliferation of mouse hepatocytes. This effect disappeared after heating the extract at 90°C for 60min, suggesting that some proteinaceous molecule is involved. We report the effect of dialysed extract (MW >12kD) on the concentration of both thyroid‐stimulating hormone (TSH) and growth hormone (GH) in an incubation medium of pituitary cells from normal and oestrogenised rats. Time‐ and dose‐dependent response of both hormones resulted in increasing TSH levels. Concentrations of GH were lower in the treated than in control pituitary cells. The time elapsed until the finding of differences suggests the presence in the mosquito extract of some protein binding the hormone. The differences were not due to lethal toxic effects since the Trypan blue viability test showed no differences between control and treated cells. Furthermore, the effect disappeared when the extract had previously been heated at 90°C for 60min. Finally, our results suggest the presence of some proteins in the mosquito Culex pipiens L. larvae, which would act as a pituitary hormone regulator.
ALTERNATIVE SPLICING OF RH BLOOD GROUP POLYPEPTIDE mRNA PRODUCES A NOVEL TRANSCRIPT CONTAINING A SHORT NUCLEOTIDE INSERTION ON HUMAN ERYTHROLEUKEMIC K562 CELLS
Authors: Sakata Noriaki , Yamazaki Kentaro , Kogure Tadahisa , Mukai Toshiji
Published: September 2002

A novel isoform of the human Rh blood group polypeptide cDNA was isolated from human erythroleukemic K562 cells. This isoform was produced by deletion of the sequences derived from exons 2 and 3 of the RHCE gene and insertion of 44‐bp into the resulting junction between exon 1 and 4 derived sequence. The deduced amino acid sequence revealed that the 44‐bp insertion sequence contains an in‐frame stop codon that causes premature chain termination. A sequence homology search using GenBank showed that the inserted sequence was derived from the intron between exons 1 and 2 of the RHCE gene. Moreover, analysis of the region surrounding the inserted sequence indicated that the insert was a cryptic exon flanked by consensus donor and acceptor splice sequences. This novel transcript was most likely produced by alternative splicing.
CARCINOGENESIS OR TUMOURIGENICITY TESTING OF ANIMAL CELL LINES FOR VACCINE PREPARATION BY COLONY FORMATION ON SOFT AGAR AND BY AGGLUTINATION UNDER PLANT LECTINS
Authors: Zhang De‐Li , Liu Shang‐Gao , Yan Long‐Fei , Li Liu‐Jin , Huang Gao‐Sheng , Fang Fu‐De , Xia Geng‐Tian , He Xu‐Yu , Gao Bu‐Xian , Bai Xiao‐Hong , Wang Wei , Ding Pei‐Guo
Published: September 2002

The carcinogenic or tumourigenic testing of seven animal kidney cell lines (F‐81, CRFK, MDCK, Vero, Vero‐2 cell line, MA‐104 and BHK‐21) established in China, were carried out in more than 700 nude mice for colony formation in soft agar and for agglutination under different density of plant lectins. Tests showed that there were correlation between cell line chromosome number variations and anchorage independence in soft agar, agglutinability under lectins and tumour‐forming ability in nude mice. Since testing in vitro was more economical, simpler and faster and thus thought to be more reliable, we recommend measuring agglutinability, followed by anchorage independence or analysis of karyotype as the initial means for monitoring tumourigenicity of animal cell lines in nude mice.
cGMP‐REGULATED STORE‐OPERATED CALCIUM ENTRY IN HUMAN HEPATOMA CELLS
Authors: Jiang J. L. , Yu M. K. , Chen Z. N. , Chan H. C.
Published: September 2002

This study aimed to investigate cGMP‐regulated store‐operated Ca2+entry in human 7721 hepatoma cells. [Ca2+]iwas measured using Fura2/AM. After incubation of the cells with 4μm thapsigargin, Ca2+entry was evoked by application of 1m m Ca2+to extracellular solution and was blocked by 3m m Ni2+, indicating the presence of store‐operated Ca2+entry in human 7721 hepatoma cell line. Application of 8‐Br‐cGMP reduced the [Ca2+]iin hepatoma 7721 cells by 80%. These data demonstrated for the first time that store‐operated Ca2+entry pathway is present in human hepatoma cells, which is regulated by cGMP.
THE EFFECT OF CYTOCHALASIN J ON KINETOCHORE STRUCTURE IN PTK1CELLS IS MITOTIC CYCLE DEPENDENT
Authors: Wrench Gregory A. , Snyder Judith A.
Published: September 2002

Mitotic PtK1cells were arrested in mitosis with nocodazole to determine the effect of cytochalasin J (CJ) on kinetochore structure in arrested and nocodazole‐released cells. In previous studies it was shown that CJ had a more pronounced effect on alteration of kinetochore structure and spindle microtubule (MT) architecture when applied during prophase or prometaphase. In this study, mitotic cells were treated at preanaphase for 10min with 1μg/ml nocodazole, or in 1μg/ml nocodazole and 10μg/ml CJ to allow for the advancement of the ‘mitotic clock’. Thus it can be determined if either changes in the timing of mitosis, the maturation of the kinetochore, and/or the lack of MT connection to the kinetochore affects the ability of CJ to detach or alter the attachment of chromosomes to the developing spindle. Preanaphase cells treated with 1μg/ml nocodazole for 10min and released into 10μg/ml CJ showed significant changes in MT organization and kinetochore structure. MTs nucleated at the centrosome are fragmented and kinetochore structure was significantly altered showing only two laminae with few MTs inserted into this structure. Preanaphase cells treated with 1μg/ml nocodazole and 10μg/ml CJ for 10min and released into 10μg/ml CJ showed similar, but more pronounced, effects on kinetochore structure and spindle MT organization. We interpret these results to suggest that CJ treatment has a greater effect on MT attachment and kinetochore structure in nocodazole pre‐treated cells, where the kinetochore structure is mature and the mitotic cycle has been advanced.
ULTRADIAN RHYTHMS AND THE lux‐GENE REPORTER SYSTEM
Authors: Šimkus R. , Meškien≐ R. , Meškys R.
Published: September 2002

‘Noisy’ bioluminescence of an unstirred and exposed‐to‐air microculture of lux ‐gene fused Escherichia coli was interpreted, assuming two oscillatory processes: cyclic induction of the lac promoter, resulting in oscillatory reporter lux ‐gene expression, and oscillatory respiration in the culture. Biological support for the theoretical argument, that two rhythms can interact in cells to produce additional rhythms, is provided. These results, along with previously obtained data on oscillatory bioluminescence, indicate the deterministic nature of observed noise‐like fluctuations and promising perspectives of lux gene reporter systems in investigations of ultradian rhythm.
[Ca2+]iRISE IN JURKAT E6‐1 CELL LINES FROM DIFFERENT SOURCES AS A RESPONSE TO 50Hz MAGNETIC FIELD EXPOSURE IS A REPRODUCIBLE EFFECT AND INDEPENDENT OF POLY‐l‐LYSINE TREATMENT
Authors: Mattsson Mats‐Olof , Lindström Ewa , Still Maria , Lindström Per , Mild Hansson Kjell , Lundgren Erik
Published: September 2002

Jurkat E6‐1 cells obtained from three different sources were compared with respect to intracellular calcium response to a 50Hz, 0.15mT, magnetic field, to treatment with poly‐l‐lysine and to protein expression at the cell surface. The fura‐2 single cell measurements were a replication study performed by three members of our group. The cells responded to the applied magnetic fields, although the percentage of responding cells was lower than in earlier studies. The geomagnetic field was backed off without changing the outcome of the intracellular calcium measurements. Fluorometric analyses showed no difference between the E6‐1 cells obtained from three sources with respect to the expression of cell surface marker molecules. The addition of the cell adhesive peptide, poly‐l‐lysine, did not itself cause any effects on the intracellular calcium concentration.
GROWTH INHIBITION, MORPHOLOGICAL DIFFERENTIATION AND STIMULATION OF SURVIVAL IN NEURONAL CELL TYPE (Neuro‐2a) TREATED WITH TROPHIC MOLECULES
Authors: Blanco Victor , Camelo Jorge Lopez , Carri Néstor Gabriel
Published: September 2002

Trophic molecules are key regulators of survival, growth and differentiation of neural cells. Neuronal cell type Neuro‐2a is a good model to study development and molecules modulating this process, and retinoic acid (RA) and neurotrophins (NGF, BDNF, NT‐3 and NT‐4) have been shown to be active in this modulation. The purpose of the present study was the functional analysis of these trophic molecules in our short‐term bioassay of Neuro‐2a cells, an immortalised murine neuroblastoma cell line. Through cell counting, image process and arithmetic combination of digital parameters of treated and untreated cultures, we show that RA inhibits growth and induces morphological neuronal phenotype of treated cells. Through DNA labelling with BrdU we also show that NGF, BDNF, and NT‐3 increase survival and proliferation of cells, grown in serum‐deprived media. From these results we conclude that neurotrophins have manifest trophic effects on cells improving survival, growth and proliferation and we also confirm the growth arrest and differentiation properties of RA on Neuro‐2a cells.