bluuebunny/crossref_metadata_2025 huggingface dataset:
NOVEL MARKERS FOR CONSTITUTIVE SECRETION USED TO SHOW THAT TISSUE PLASMINOGEN ACTIVATOR IS SORTED TO THE REGULATED PATHWAY IN TRANSFECTED PC12 CELLS
Authors: HARRISON T. M. , CHIDGEY M. A. J. , UFF S.
Published: October 2002

The rat pheochromocytoma cell line PC12 contains two distinct pathways of protein secretion. Proteins secreted via the regulated pathway are stored in secretory vesicles and exocytosed only in response to a specific signal, whereas proteins secreted via the constitutive pathway are exported continuously. Analysis of regulated secretion of a heterologous protein in this system often relies on comparison of secretion rates with those of endogenous proteins known to be secreted via the constitutive route. In order to improve these controls, we have evaluated a number of secreted enzymes, selected for the sensitivity and convenience of their assays, as transgenic markers for the constitutive pathway. We show that both human‐secreted placental alkaline phosphatase (SEAP) and bacterial β‐lactamase operate in this way in transfected PC12 cells. In contrast, transfected human tissue plasminogen activator (tPA) is shown to be sorted to the regulated pathway.
SYNERGISTIC REGULATORY EFFECTS OF TNFα, IL‐1αAND IFNα ON THE GROWTH AND DIFFERENTIATION OF DAUDI LYMPHOMA CELLS
Authors: SANTAVENERE E. , DI PIETRO R. , CENTURIONE M. A. , DI DOMENICANTONIO L. , TRUBIANI O. , RANA R. A.
Published: October 2002

The regulatory effects of the combined treatment of tumour necrosis factorα (TNFα), interleukin‐1α (IL‐1α) and interferonα(IFNα) on the growth and differentiation of Daudi lymphoma cells were investigated. By means of anti‐BrdU monoclonal antibodies and [3H‐thymidine] incorporation a reduced proliferation rate was shown both through a combi‐nation of TNFα with either IL‐1α or IFNα and, above all, through simultaneous treatment with the three cytokines. In parallel, the degree of differentiation was evaluated via morphological criteria and detection of Fc receptors (FcR) and appeared higher after treatment with the three cytokines. Our results provide evidence of the increased sensitivity of this cell line to this combined treatment supporting the existence of a synergistic interaction in inducing the antiproliferative and differentiative effects.
HUMAN SPERM β‐GLUCURONIDASE IS POORLY EXTRACTABLE BY TRITON X‐100
Authors: BRANDELLI ADRIANO , MIRANDA PATRICIA V. , TEZÓN JORGE G.
Published: October 2002

A part of sperm glycosidase activities was detected as detergent‐insoluble after sequential extractions with Triton X‐100. Sixty per cent of total β‐glucuronidase activity was found in the detergent‐insoluble fraction. This portion of β‐glucuronidase was resistant to extractions in the presence of 1mKCl, chaotropic agents, colchicine or cytochalasine B, being only partially solubilized by 3mKCl or DNAse I treatment. Results demonstrate that β‐glucuronidase is tightly associated to the Triton X‐100 resistant fraction.
THE CONFORMATION OF CLATHRIN LIGHT‐CHAINS ON TRISKELIONS PROBED BY ANTIBODY INHIBITION
Authors: JACKSON A. P.
Published: October 2002

The conformation of clathrin light‐chains along the proximal arm of the clathrin triskelion was studied by using rabbit anti‐(light‐chain peptides) to inhibit the binding of a mouse monoclonal antibody against an epitope in the amino‐terminal region. Prior incubation of triskelions with rabbit antisera raised against the extreme carboxyl‐terminal of the light‐chains partially inhibited binding. The inhibition was largely removed when tested on light‐chains that had been freed from triskelions. This suggests that when the light‐chains bind the heavy‐chain, they adopt a conformation in which the amino and carboxyl‐terminal domains are not fully extended, but fold such that these two domains face each other.
NUCLEOLAR ORGANIZATION AS REVEALED IN CELL SPREADS
Authors: GHOSH SIBDAS , PAWELETZ NEIDHARD
Published: October 2002

The nucleolar organization has been studied in spreads of cells either untreated or treated with hypotonic salt solution for different periods. A network corresponding to the nucleolonema becomes evident with progressive hypotonic treatment. The network reveals units comparable to the rDNA transcriptional units in length and is associated with tufts of fibrils and granules. Spread preparations from cycloheximide treated cells reveal a thread‐like axis and often ‘Christmas tree’‐like configurations within these units. Spacers joining the units can also be detected. It is supposed that the transcriptional units move outwards with their transcriptional products where the processing takes place. In loose nucleoli, this network forms the nucleolonema, which remains associated with the granules, the processed transcriptional products. In compact nucleoli the network is obliterated by the granules and they form the major component of the nucleoli. Such organization represents all the events in the transcription and processing of ribosomal RNA.
DISTINCT, VERY HIGH FREQUENCY OSCILLATIONS IN THE ACTIVITY AND AMOUNT OF ACTIVE ISOZYME OF LACTATE DEHYDROGENASE IN MURINE ERYTHROLEUKAEMIC CELLS AND A CELL‐FREE SYSTEM
Authors: FERREIRA G. M. N. , HAMMOND K. D. , GILBERT D. A.
Published: October 2002

Electrophoretic and kinetic determinations of the activity of lactate dehydrogenase in murine erythroleukaemic (MEL) cells, sampled at 1min intervals, reveal distinct oscillations in the activity and amount of active isozyme. Both oscillations have periods in the range of 2–6min (probably less) and both appear to be rhythmically modulated with respect to period, amplitude and mean. The oscillations also occur in cell‐free systems, a fact which throws doubt on the value of studies where it is assumed that such preparations have constant composition.
RELATIONSHIP BETWEEN PINOCYTOSIS AND ADHESION IN AMOEBA PROTEUS
Authors: KŁOPOCKA W. , KOŁODZIEJCZYK J. , LOPATOWSKA A. , GREBECKI A.
Published: October 2002

Induction of pinocytosis in Amoeba proteus is independent of adhesion. It is manifested by non‐adhering floating specimens, as well as by amoebae moderately adhering and locomoting on the glass, or tightly attached to the polylysine‐coated substratum. The formation of pinocytotic rosettes results in de‐adhesion, at the beginning of pinocytosis on the glass, but at its end on the polylysine. It suggests an opposition between adhesion and cell shape transformation. Pinocytosis and adhesion are both inhibited, by an unknown mechanism, in the presence of gelatin either in the substratum or in solution.
ALBUMIN CAN REVERSE THE RELEASE OF POTASSIUM FROM HUMAN ERYTHROCYTES TREATED WITH THE NON‐IONIC DETERGENT, BRIJ 58
Authors: BOGNER PETER , WHEATLEY DENYS N. , BORBÉLY CSABA , MISETA ATTILA
Published: October 2002

Exchange of erythrocyte intracellular (i/c) K+for extracellular (e/c) Na+in human erythrocytes treated with sub‐CMC concentrations of the non‐ionic detergent Brij 58 can be stopped by reincubation in serum or albumin containing solutions. The progressive equilibration of the K+contents of detergent‐treated human erythrocytes with the incubation medium was reversed by an albumin‐mediated withdrawal of detergent molecules from the cell. Re‐establishment of near normal [K+] in terms of K+/kg water proceeds in two ways: (i) a metabolism‐dependent net accumulation of K+ions; and (ii) a metabolism‐independent shrinkage of erythrocytes, this being the more significant factor.
PROPERTIES OF NUCLEAR MATRIX PROTEINS THAT BIND THE 5′ FLANKING REGION OF THE HAPTOGLOBIN GENE ARE CHANGED DURING THE ACUTE‐PHASE RESPONSE
Authors: POZNANOVIĆ GORAN , GRUJIĆ VESNA , IVANOVIĆ‐MATIĆ SVETLANA , ŠEKULARAC SNEŽANA
Published: October 2002

It was previously shown that during the acute‐phase response‐induced elevated transcription of the rat haptoglobin gene, protein p55 and the lamins mediate the increased binding of restriction fragment II (−541/−146) via a 38 bp adenine tract lying 147bp upstream from the haptoglobin gene cis element (−165/−56), to scaffold type II‐like nuclear matrices. Here we show that the fragment II binding pattern to ≥40 kDa proteins of nuclear matrices analogous to type I scaffolds is more complex. Fragment II bound conspicuously to a 40 and less so to p55, a 60 kDa protein and the lamins of control matrices. During increased gene transcription, it bound prominently to p55, the lamins, and less so to 45 and 52 kDa proteins. This was accompanied by the tenacious binding of the DNA to isolated nuclear matrices in vitro and post‐translational modifications of certain matrix proteins. The lamins and p55 demonstrated a greater N‐acetylglucosamine/sialic acid content and p55 an increased in vitro phosphorylation by a nuclear matrix‐associated cyclic‐nucleotide‐independent kinase. The acute‐phase response also caused an increased partitioning of p55 with the nuclear matrix. It was concluded that, as a result of a molecular remodelling of the nuclear matrix at the point of contact with chromatin, the nature of its association with region II DNA changed during elevated haptoglobin gene expression.
SYNERGISTIC ANTITUMOUR ACTIVITY OF VITAMINS C AND K3AGAINST HUMAN PROSTATE CARCINOMA CELL LINES
Authors: VENUGOPAL MEENAKSHI , JAMISON JAMES M. , GILLOTEAUX JACQUES , KOCH JOHN A. , SUMMERS MARCIA , HOKE JASON , SOWICK COLLEEN , SUMMERS JACK L.
Published: October 2002

Vitamins C, K3(VC, VK3) and a VC/VK3combination with a VC:VK3ratio of 100:1 were assayed for their antitumour activity against two human prostatic carcinoma cell lines. Co‐administration of the vitamins enhanced the antitumour activity 5‐ to 20‐fold even with a 1 h exposure time. While exogenous catalase destroyed the antitumour activity, hydrogen peroxide‐induced lipid peroxidation was negligible. Analysis of cellular ATP and thiol levels as well as DNA and protein synthesis revealed: a transient increase in ATP production, a decrease in DNA synthesis, an increase in protein synthesis and a decrease in thiol levels. These results suggested that the increased cytotoxicity of the vitamin combination was due to redox cycling and increased oxidative stress.
REGULATION OF ION TRANSPORT BY P2UPURINOCEPTORS AND α2A ADRENOCEPTORS IN HT29 CELLS
Authors: ZHANG WEI , ROOMANS GODFRIED M.
Published: October 2002

X‐ray microanalysis was applied to investigate ion transport mediated by P2Upurinoceptors and α2A adrenoceptors as well as their interaction in the regulation of the intracellular elemental concentration in HT29 cells. In response to ATP, HT29 cells showed a decrease of intracellular Cl, Na and an increase in Ca. A similar result was observed with UTP, but UTP appeared to be more potent than ATP. On the other hand, UK14,304, an α2receptor agonist, was found to be capable of reversing the action of both UTP and ATP on ion secretion, and caused a clear increase in intracellular Na and Cl. Moreover, treatment of cells with UK14,304 before exposure to UTP did not induce a decrease in Cl and Na, suggesting that UK14,304 blocks the action of UTP. The secretory effect of UTP was also blocked by NPPB, a chloride channel blocker, and alloxan. Chelation of extracellular Ca with EGTA abolished ion response to UTP. These results suggest that since inhibition of the intracellular cAMP system and chelation of Ca2+can block the nucleotide‐induced chloride secretion, the ATP and UTP‐induced chloride secretion can be mediated via both cAMP‐dependent and Ca2+‐dependent pathways.
A RAPID BIOASSAY TO DETECT MYCOTOXINS USING A MELANIN PRECURSOR OVERPRODUCER MUTANT OF THE CILIATETETRAHYMENA THERMOPHILA
Authors: GONZÁLEZ ANA MARTÍN , BENÍTEZ LAURA , SOTO TRINIDAD , DE LECEA RODRIGUEZ JOSEFINA , GUTÍERREZ JUAN CARLOS
Published: October 2002

Four different mycotoxins (patulin, T‐2 toxin, diacetoxyscirpenol and roquefortine) were used to study growth inhibitory effects on a melanin precursor overproducer mutant of the ciliateTetrahymena thermophilaThis strain is especially sensitive to diacetoxyscirpenol and T‐2 toxin. The secretion capacity of melanin precursors into the culture medium by this mutant and its biosensor capacity are very useful characteristics to elaborate a rapid bioassay to detect some specific mycotoxins.
TREATMENT OF RAT PROXIMAL AND DISTAL COLONIC CELLS WITH SODIUM ORTHOVANADATE ENHANCES THEIR ADHESION AND SURVIVAL IN PRIMARY CULTURE
Authors: KAEFFER B. , BÉNARD C. , BLOTTIÈRE H.M , CHERBUT C.
Published: October 2002

We have studied the effect of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on primary cultures of colonocytes and stromal cells. Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37°C to prepare viable gland fragments and isolated cells. Cell preparations were inoculated onto plastic substratum or cytodex‐3 microcarriers in a defined maintenance medium or in 1% fetal calf serum media. Incorporation of sodium orthovanadate (≥50 μm) in these media constantly enhanced the survival (cell enumeration and trypan blue exclusionP<0.05) and the adhesion (up to four‐fold by crystal violet staining,P<0.01) of colonocytes (characterized by cytokeratin‐18, transforming growth factor‐α or alkaline phosphatase expression) and stromal cells. Removal of sodium orthovanadate from culture media restored cellular death processes. Incorporation of 10 mmn‐butyric acid did not promote cell adhesion and survival except for distal cells exposed to 2 mm sodium orthovanadate. Besides studies in the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non‐cancerous colonic cells.
MAPPING OF G2/M‐PHASE PREVALENCES OF CHAPERON‐ENCODING TRANSCRIPTS BY MEANS OF A SENSITIVE DIFFERENTIAL HYBRIDIZATION APPROACH
Authors: DITTMAR GUNNAR , SCHMIDT GEBHARD , KOPUN MARIJANA , WERNER DIETER
Published: October 2002

The sensitivity of the differential hybridization approach is significantly increased by the application of size‐selected probes. RNA from elutriated phase‐synchronous Ehrlich ascites tumor (EAT) cells has previously been used to prepare cell cycle phase‐specific cDNA libraries in the in‐vitro transcription vector pBluescript. PCR amplification of the libraries with vector‐fitting primer pairs generates amplified cDNA reflecting the mRNA complexities of cells in G1, S and G2/M phases. Probes with reduced complexities were recovered after side‐by‐side electrophoresis of equal amounts of PCR‐amplified cDNA and elution of probes from parallel gel sections. Such size‐selected probes release significant differential clones which escape their detection in the conventional differential hybridization approach. Three clones hybridizing preferentially with the G2/M phase‐specific probe were further characterized. The genes were identified by their nucleotide sequences. They encode proteins known to be involved in protein folding: heatshock cognate protein, HSC 70; heatshock cognate protein, HSC 73; η subunit of the chaperonin containing TCP‐1 complex, CCT. The G2/M phase‐prevalent expression of these genes were further verified on the mRNA and on the protein level by Northern and Western blot analysis which confirms the significance of the differential hybridization approach and which indicates that the expression of this group of proteins increases with cell cycle progression. The expression of the chaperonin‐containing TCP‐1 complex appears to be specifically linked with the S to G2/M phase transition of the cell cycle.
IL‐1 MODIFIES SYSTEM A TRANSPORT ACTIVITY IN HUMAN RHEUMATOID SYNOVIAL CELLS
Authors: LE MAIRE VALÉRIE , HERNVANN ALAIN , LE MARECHAL HERVÉ , EKINDJIAN OHVANESSE G. , AUSSEL CHRISTIAN
Published: October 2002

Given the importance of interleukin‐1 in both rheumatic diseases and the modulation of cell metabolic activities, we studied the action of this cytokine on the neutral amino acid transport A system on rheumatoid synovial cells. In these cells IL‐1 (1ng/ml) induced amino transport stimulation from 5min to 5h. This effect was obtained only after a starvation period. No concentration‐related effect was found for IL‐1‐stimulated MeAIB uptake, and the IL‐1‐mediated MeAIB uptake stimulation is independent of protein synthesis. Neosynthesis or post‐translational maturation of protein transport is a prerequisite for obtaining this effect. In conclusion, rheumatoid synovial cells exhibit a higher sensitivity for IL‐1 than osteoarthritic ones, probably related to their intense metabolic activity.
THE EFFECT OF ENVIRONMENTAL ANEUPLOIDY‐INDUCING AGENTS ON THE MICROTUBULE ARCHITECTURE OF MITOTIC MERISTEMATIC ROOT CELLS INHORDEUM VULGARE
Authors: VOUTSINAS G. , ZARANI F.E , KAPPAS A.
Published: October 2002

Microtubules are the prime components involved in chromosomal segregation, their functional accuracy ensuring maintenance of the normal karyotype in the progeny. Chemically‐induced disruption of microtubules during mitosis can lead to aneuploidy. In this study, seven environmental chemicals, i.e. cadmium chloride (CD), econazole nitrate (EZ), benomyl (BM), thiabendazole (TB), griseofulvin (GF), thimerosal (TM) and hydroquinone (HQ), were tested for their ability to induce microtubule disruption in mitotic meristematic root cells of the higher plantHordeum vulgarewith the use of anti‐tubulin indirect immunofluorescence microscopy. All chemicals tested in this study, with the exception of TB and HQ, produced modifications in the morphology of microtubule organization and reduced the fidelity of the spindle apparatus inHordeum vulgare
INDUCTION OF KERATINOCYTE PROLIFERATION BY A SHORT TREATMENT WITH KERATINOCYTE‐CONDITIONED MEDIUM
Authors: PAINI C. , SAVANT F. , MALCOVATI M. , TENCHINI M. L.
Published: October 2002

The effect of a short treatment with keratinocyte‐conditioned medium (KCM) on the growth of normal human epidermal keratinocytes was investigated. Serum‐free MCDB153 medium was conditioned by keratinocytes for 24h after plating. Following attachment to uncoated plastic surfaces (4h after plating), cells were exposed for 20h to KCM. After 10 days of culture in MCDB153 medium, an increase of about six‐fold in cell number was observed in KCM‐treated plates over controls, indicating that a short treatment with KCM is sufficient to induce cell proliferation. The effect of addition of KCM at different times after plating was also evaluated: KCM treatment resulted to exert its maximum effect on cell proliferation, when performed immediately after the completion of attachment of cells to the surface of the dish. Mitogenic activity present in KCM is not inhibited by heparin sulphate. The kinetics of accumulation of this early secreted growth‐stimulating activity showed that a plateau is reached within 24h of conditioning. These data suggest that this mitogenic activity should not be amphiregulin. The observation that, following KCM treatment, the majority of cells is able to incorporate [3H]‐thymidine as compared to controls suggests that the observed final increase in cell number is due to an increase in the number of cycling cells rather than to a shortening of the cell cycle.
MAJOR LAMININ‐BINDING AND F‐ACTIN‐LINKED GLYCOPROTEINS OF NEUTROPHILS
Authors: KERESZTES MARGIT , LAJTOS ZSUZSA
Published: October 2002

Two major EHS‐laminin‐binding membrane glycoproteins—with apparent molecular masses of 50kD and 18kD—were shown by protein blotting in membrane fractions of porcine neutrophils. These galectin‐like glycoproteins (binding probably via the N‐acetyllactosamine sequences to laminin) could also be detected by labelled F‐actin in protein blots. Following 35min adhesion to the plastic surface, the relative amount of the 18kD protein increased considerably in the light (plasma membrane) and in the dense (intracellular) membrane fractions of the attached cells; the 50kD polypeptide (identified as a CD14‐like protein) seemed to accumulate characteristically in the dense membrane fraction. These observations imply that direct connections could be formed between membrane glycoproteins and microfilaments during cell—substrate adhesion which may be preceded by enhanced cell surface targeting of certain adhesion receptors.
MOLECULAR CLONING OF THE BOVINE α1(IV) PROCOLLAGEN GENE (COL4A1) AND ITS USE IN INVESTIGATING THE REGULATION OF EXPRESSION OF TYPE IV PROCOLLAGEN BY RETINOIC ACID IN BOVINE LENS EPITHELIAL CELLS
Authors: SAWHNEY RAJINDER S. , WOOD LINDA S. , VOGELI GABRIEL
Published: October 2002

This report is the first to describe the isolation of a 400 base pair cDNA clone encoding part of the bovine α1(IV) procollagen. Using the polymerase chain reaction (PCR), we have amplified a sequence of approximately 400bp from this gene within the recombinant phage DNA. The cloned sequence encodes 94 amino acids that form part of the protein's helical region. The sequence contains one interruption in the Gly‐Xaa‐Yaa repeat unit. The third base of the codon for glycine at several sites differs from those seen in murine and human genes, as does the third base of proline codons. The bovine cDNA also contains fewer thymine residues. Northern blot hybridization has shown that the mRNA for bovine procollagen to be 6.2kb in size. We have used the cDNA clone to investigate the effect of all‐transretinoic acid (RA) on the gene expression of α1(IV) procollagen in cultured bovine lens epithelial (LE) cells. We have also observed that RA decreases total protein production and concomitantly increases type IV procollagen in a concentration dependent manner. An increase in α1(IV)mRNA as well as increase in type IV procollagen suggest that the regulation of α1(IV) gene by RA in the LE cells is at the transcriptional level. Further, our results support the hypothesis that RA inhibition of lens epithelium transformation to fibroblast‐like cells may be due to the ability of RA to stimulate the production of basement membrane components by epithelia.
ROLB EXPRESSION PATTERN IN THE EARLY STAGES OF CARROT SOMATIC EMBRYOGENESIS
Authors: DI COLA ALESSANDRA , POMA ANNA , SPANÒ LAURA
Published: October 2002

The pattern of expression of therolBgene, derived from the T‐DNA of the plant pathogenAgrobacterium rhizogenes, has been investigated during the early stages of somatic embryo formation in suspension cultures of carrot (Daucus carotaL.). The reporter geneGUS(Escherichia coliβ‐glucuronidase), under transcriptional control of full‐lengthrolB promoter region, has been utilized in order to evaluate both qualitative and quantitative variations in the expression pattern. Fluorimetric measurements point to the developmental regulation of the gene, while results from histochemical analysis indicate that the promoter ofrolBis firstly activated in the central (core) region of the globular embryos.
CELL—CELL CONTACTS ACCELERATE CELL COLONY GROWTH IN SPARSE CULTURES OF CHICK EMBRYO FIBROBLASTS
Authors: GASPARIAN G. , SARKISSIAN N. , SAYADIAN A. , GRIGORIAN R.
Published: October 2002

There is no cell proliferation in very sparcely plated chick embryo cell cultures. Substituting conditioned medium or adding of ethanol‐fixed homologous cells to the cultures accelerates cell colony growth. The mechanism for the mitogenic action of fixed cells is considered to be the contact stimulation of cell proliferation, and addition of extra cells to sparse culture is believed to mimic the cell micro‐environment existing in subconfluent cultures. The role of diverse cell—cell contacts in cultured cell growth regulation is discussed. The procedure used (addition of ethanol‐fixed cells) may improve normal cell cloning techniques.
HISTONES AS EXTRACELLULAR MESSENGERS: EFFECTS ON GROWTH HORMONE SECRETION
Authors: BROWN O. A. , SOSA Y. E. , GOYA R. G.
Published: October 2002

Histones display hormone‐like properties when present in extracellular fluids. The authors report that histones H2A and H2B possess growth hormone (GH)‐releasing activityin vitroand describe the specificity and signal transduction pathways involved in these effects. Perfused and incubated rat pituitary cells were used in different sets of experiments and GH release was measured by radio‐immunoassay (RIA). Perfusion of cells with 30μmhistone H2A or H2B, generated significant GH secretory responses. Cells incubated with histone H2A showed a dose‐ and time‐dependent stimulatory effect on GH release which was blocked by peptide MB35, a synthetic fragment of histone H2A. Incubation of pituitary cells with the GH secretagogue GHRP‐6, and histones revealed an additive release of GH, whereas GHRH and histones revealed a synergistic effect. The basic peptide poly‐Lys did not mimetize the action of histones. Both EGTA and the protein kinase C inhibitor trifluoperazine, but not the calcium ionophre A23187, were able to reduce significantly the GH response of somatotrophs to histones. Pituitary cell incubation with 30μmforskolin alone or in the presence of H2A or H2B, stimulated GH release in the same magnitude. The results confirm previous evidence that histones may act as hypophysotropic signals and suggest, although do not prove, that this activity is receptor dependent. Calcium‐ and diacylglycerol‐associated pathways participate in these effects.
DECREASED DENSITIES OF INTRAMEMBRANOUS PARTICLES AND CYTOCHEMICALLY DETECTABLE CHOLESTEROL IN MICROVILLI OF STARVED RAT ENTEROCYTES
Authors: WAHEED A. A. , TOYAMA Y. , YASUZUMI F. , GUPTA P. D.
Published: October 2002

The densities of intramembranous particles (IMPs) and of sterol complexes induced by treatment of filipin were studied by freeze‐fracture replication of intact intestine and/or isolated brush border membranes (BBM) of well‐fed and starved rats. The density of IMPs and filipin‐sterol complexes (FSCs) decrease considerably during starvation. Biochemical estimations show a decrease in the levels of cholesterol and proteins with respect to phospholipids during starvation which is in agreement with morphological findings. It is suggested that these changes may play a role in regulating membrane fluidity which in turn affects absorption of nutrients through BBM.
CELL POSITION‐DEPENDENT RECIPROCAL FEEDBACK REGULATION OF TYPE I COLLAGEN GENE EXPRESSION IN CULTURED HUMAN SKIN FIBROBLASTS
Authors: HATA RYU‐ICHIRO , AKAI JUN , KIMURA AKINORI
Published: October 2002

In order to investigate possible cell positional effects on the gene expression of human dermal fibroblasts, the authors cultured the cells on non‐coated polystyrene culture dishes, type I collagen‐coated dishes, or collagen gels formed by type I collagen, or suspended them in type I collagen gels and measured collagen synthesis by the cells. The production rate of type I collagen was similar whether cells were cultured on non‐coated polystyrene or on type I collagen‐coated dishes, but it was suppressed significantly when the cells were placed within the collagen gel matrix. Time‐dependent expression of genes for α1(I) and α2(I) collagen chains was measured by Northern blot analysis. A significant increase in mRNA levels for these chains was observed when the cells were cultured for three days on type I collagen‐coated dishes or on collagen gels. On the other hand, a significant decrease in the mRNA levels was observed after 2 days and later, when the cells were cultured within type I collagen gel matrix. These results indicate that human dermal fibroblasts recognize their position on or in type I collagen (extracellular matrix) and respond by changing their expression patterns of type I collagen chain genes. The results of the kinetics of gene expression also suggest that upregulation and downregulation of type I collagen genes are controlled by different mechanisms.
LOCALIZATION AND DISTRIBUTION OF PITUITARY ADENYLATE CYCLASE‐ACTIVATING POLYPEPTIDE IN THE RAT EPIDIDYMIS
Authors: LEUNG P. S. , WONG T. P. , WONG P. Y. D. , CHAN H.C
Published: October 2002

Previous investigation has provided evidence for the control of electrogenic chloride secretion by pituitary adenylate cyclase‐activating polypeptide (PACAP) across the rat epididymal epithelium using electrophysiological measurement of transepithelial transport in cultured epididymal system. Hence, it suggests that epididymal and sperm functions are subject to control by a local PACAP system in the rat epididymis. In the present study, localization and distribution of PACAP in the rat epididymal duct was studied by an indirect immunofluorescence technique in conjunction with confocal laser scanning microscopy. Immunoreactivity for PACAP was found in all regions of the epididymal duct. However, the intensity of immunoreactivity for PACAP was stronger in the caput and corpus regions when compared to that of the cauda epididymidis. Much weaker immunostaining for PACAP, as compared to those found in other regions, was observed in the cauda epididymidal tubules which are in close proximity to the vas deferens. No immunoreactivity for PACAP was found in epididymal spermatozoa. Together with the previous finding, the present results suggest that PACAP may exhibit a regional difference in its expression along the epididymal duct and it may act in a paracrine or autocrine fashion in the regulation of epididymal chloride secretion and hence fluid secretion, thus regulating epididymal and sperm functions along the epididymal duct.
ALTERATIONS IN THE GROWTH AND ADHESION PATTERN OF VERO CELLS INDUCED BY NUTRITIONAL STRESS CONDITIONS
Authors: GENARI SELMA CANDELÁRIA , GOMES LAURECIR , WADA MARIA LÚCIA FURLAN
Published: October 2002

The pattern of growth, adhesion and protein synthesis in Vero cells submitted to nutritional stress conditions was investigated. The control cells presented a characteristic pattern, with monolayer growth, while the stressed cells presented multilayered growth, with aggregate or spheroid formation which detached on the flask surface and continued their growth in another region. In the soft agar assay, with reduced amount of nutrients, only the stressed cells presented growth, indicating physical and nutritional independence. A 44‐kDa protein was observed in stressed cells and was absent in non‐stressed cells. The adhesion index and fibronectin synthesis and distribution were altered in stressed cells. After confluence, control cells presented fibronectin accumulation in lateral cell—cell contact regions, while this fibronectin accumulation pattern was not observed in stressed cells. These alterations may be responsible for the multilayered growth and decreased adhesion index observed in stressed cells which were transformed by nutritional stress conditions.
THE OLIGOMERIC INTEGRITY OF TOPOSOME IS ESSENTIAL FOR ITS MORPHOGENETIC FUNCTION
Authors: SCATURRO GIOVANNI , ZITO FRANCESCA , MATRANGA VALERIA
Published: October 2002

Sea urchin embryos are uniquely suitable for the study of morphogenetic cell interactions. Efforts to identify the molecules responsible for morphogenetic cell adhesion led to the isolation of a 22S glycoprotein complex from Paracentrotus lividus sea urchin embryo, that has been called toposome. The biological activity of toposome in mediating cellular adhesion has been fully documented. Its function in determining positional guidance during the development of the sea urchin embryo has been proposed. Here studies on the molecular structure of toposome are reported showing that, under non‐reducing conditions, it is resolved in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) in a major band with an apparent molecular weight of 260 kDa, a doublet of 180–160kDa and a lower band of 80kDa. Digestion with EndoH endoglycosidase reduced the molecular sizes of the bands of 10%, 20% and 40%, respectively. In order to establish if the oligomeric integrity of toposome was essential for its function, the biological activity of each subunit on cells dissociated from sea urchin blastula embryos was tested. The resulting swimming embryoids were lacking skeleton, while reaggregating cells supplemented with native toposome developed into pluteus‐like structures with skeletal elements.
CYTOSKELETAL CHANGES IN PLATELETS INDUCED BY THROMBIN AND PHORBOL MYRISTATE ACETATE (PMA)
Authors: CHEN RIYAN , LIANG NIANCI
Published: October 2002

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin‐treated,32P‐prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton‐insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS‐PAGE and autoradiography. Incorporation of actin into the Triton‐insoluble fraction was used to quantify the formation of F‐actin. Thrombin‐stimulated platelet cytoskeletal composition was different from PMA‐stimulated cytoskeletal composition. Thrombin‐stimulated platelets contained not only the three major proteins: actin (43kDa), myosin (200kDa) and an actin‐binding protein (250kDa), but three additional proteins of Mr56kDa, 80kDa and 85kDa in the cytoskeleton, which were induced in by thrombin dose—response relationship. In contrast, PMA‐stimulated platelets only induced actin assembly, and the 56kDa, 80kDa and 85kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56kDa, 80kDa and 85kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin‐activated platelets aggregation could involve additional cytoskeletal components (56kDa, 80kDa, 85kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.
EFFECTS OF SOME BIOLOGICALLY ACTIVE COMPOUNDS ON PHAGOSOME—LYSOSOME FUSION IN PERITONEAL MACROPHAGES OF MICE
Authors: MOZHENOK TAMARA , BELYAEVA TATYANA , BULYCHEV ANATOLY , KUZNETSOVA IRINA , LEONTIEVA EKATERINA , FADDEJEVA MARIA
Published: October 2002

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome‐lysome (P—L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P—L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA‐DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F‐actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA‐DPH polarization, FR increases DPH and TMA‐DPH polarization, and CR causes only an increase in TMA‐DPH polarization in lysosomal membranes. All three compounds tested increase F‐actin content in peritoneal macrophages. Thus, the effect of BR on P—L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P—L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.
REGULATION OF RETINOID X RECEPTOR‐γ GENE TRANSCRIPT LEVELS IN RAT HEART CELLS
Authors: GEORGIADES PANTELIS , BRICKELL PAUL M.
Published: October 2002

Retinoid X receptor‐γ (RXRγ) is a transcription factor that mediates retinoid signalling and is expressed in rat heart during adult life. However, its expression in embryonic and neonatal heart has not been investigated and it is not known whether ventricular cardiomyocytes express RXRγ or whether all‐ trans ‐retinoic acid (tRA) and thyroid hormone (T3) could influence RXRγ transcript levels in these cells. First, in situ hybridization experiments were used to test for any spatio‐temporal correlation between RXRγ gene expression and the previously shown requirement for retinoid signalling in embryonic ventricular cardiomyocytes. It was shown that RXRγ transcripts are not detectable in embryonic heart at all developmental stages examined under conditions where they are detectable in other embryonic tissues. Second, Northern blotting was used to examine whether there is a difference in RXRγ transcript levels between neonatal and adult heart. We show that levels of two RXRγ transcripts are developmentally regulated during the postnatal period because they differ between neonatal and adult hearts. Third, it was demonstrated that primary cultures of neonatal rat ventricular cardiomyocytes express RXRγ transcripts, making them a novel in vitro system for the study of RXRγ gene regulation in heart‐derived cells. Finally, this system was used to examine whether tRA and T3can influence levels of RXRγ transcripts because they have been shown to have antagonistic effects in this system and to influence RXRγ RNA levels in other systems. It was shown by Northern blot experiments, that in this system, RXRγ transcript levels are differentially influenced by these two hormones. The significance of these findings in relation to previously published work is discussed.
DIFFERENTIAL ALTERATIONS IN THE DISTRIBUTION OF THREE PHOSPHATASE ENZYMES DURING THE PLASMA MEMBRANE TRANSFORMATION OF UTERINE EPITHELIAL CELLS IN THE RAT
Authors: Bucci Maria , Murphy Christopher R.
Published: October 2002

Ultrastructural and light microscopic cytochemical methods were used to study the distribution and changes in distribution of three phosphatase enzymes: 5′‐nucleotidase (5N); thiamine pyrophosphatase (TPP); and adenosine triphosphatase (ATP) in the rat endometrium during early pregnancy up to the time of blastocyst attachment. The authors were particularly interested in changes in the apical plasma membrane and reaction product for all three enzymes was clearly localized along this membrane especially on day 1 of pregnancy. However, the three enzymes showed markedly different patterns of organization of reaction product at later times during early pregnancy. 5N, while showing a continuous lining along the microvilli on day 1 was virtually undetectable by day 6. TPP was also strongly present apically on day 1, but reaction product was not always found as a continuous lining. Again, by day 6, there was no presence of this enzyme along the apical surface. ATP differed from the other two in that it produced a strong, and relatively unchanged reaction product along the apical plasma membrane from day 1 through to day 6 of pregnancy. The changes in distribution of these enzymes was particularly obvious at the electron microscopic level and we consider their contribution to the process of ‘plasma membrane transformation’ of early pregnancy.
Migratory phenotypes of HSC‐3 squamous carcinoma cell line induced by EGF and PMA: Relevance to migration of loosening of adhesion and vinculin‐associated focal contacts with prominent filopodia
Authors: Kawahara Ei , Tokuda Ryouko , Nakanishi Isao
Published: October 2002

Cell migration is involved in carcinoma cell invasion and wound healing. We examined motogenic cytokines that potentiated migration of human HSC‐3 carcinoma cells. To assess migratory activity, modified Boyden chambers were used. Among a variety of potential motogenic cytokines, epidermal growth factor (EGF) enhanced migration of HSC‐3 cells both on collagen and fibronectin. Phorbol myristate acetate (PMA) also enhanced migration. Inhibitors of protein kinase C completely inhibited PMA‐induced migration, but only partly inhibited EGF‐induced migration. Protein kinase A was also involved in the EGF‐induced signaling pathway for migration. Although the signaling pathways were independent, and the cell shape on collagen was different from that on fibronectin, migratory cells stimulated by EGF or PMA showed common morphology on different ligands. The cells were polygonal or round in shape and the loss of long cytoplasmic extensions was noted. Migratory HSC‐3 cells stimulated by EGF or PMA became less adhesive to collagen and fibronectin. Since both EGF‐ and PMA‐stimulated migration did not require de novo protein synthesis, the signaling pathways possibly lead to assembly and disassembly of an actin cytoskeleton. Immunofluorescence for vinculin was concentrated into focal contacts in EGF‐ and PMA‐stimulated HSC‐3 cells, whereas the fluorescence signal was hardly detected in non‐stimulated cells. Talin and β1 integrin were immunolocalized at focal contacts in non‐stimulated cells, and it remained unchanged in stimulated cells. Numerous filopodia visualized with actin immunofluorescence were formed around stimulated HSC‐3 cells, whereas filopodia were short and sparse around elongated cytoplasms in non‐stimulated cells. Thus, shortening of cytoplasmic extensions with numerous filopodia, loosening of adhesion, and vinculin‐associated focal contacts were regarded as migratory phenotypes.
Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts
Authors: Gogly B. , Dridi M. , Hornebeck W. , Bonnefoix M. , Godeau G. , Pellat B.
Published: October 2002

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1–400μg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi‐linearly with increasing doses of heparins from 1 to 400μg/ml. TIMP‐1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1–10μg/ml, heparins stimulated the secretion of both 72‐kDa gelatinase (1.4–1.6‐fold) and particularly TIMP‐2 (>4‐fold). At high doses, MMP‐2 and TIMP‐2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.
SPECTRIN‐LIKE PROTEINS IN GREEN ALGAE (DESMIDIACEAE)
Authors: Holzinger Andreas , DE Ruijter Norbert , Emons Anne Mie , LüTz‐Meindl Ursula
Published: October 2002

Immunochemical detection of actin as well as spectrin‐like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross‐reactivity with desmid proteins is found at about the molecular mass of 220kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120kDa and 70kDa, which are probably breakdown products. An antibody against chicken α‐actinin, a small protein of the spectrin superfamily, recognizes bands at 90kDa, where it is expected, and 70kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI4.6 with antibodies against spectrin. Immunogold labelling of spectrin and α‐actinin antigens on high‐pressure frozen, freeze‐substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin‐like proteins in desmid cells which are probably functional in exocytosis.
MICROFILAMENT‐DEPENDENT MODULATION OF CYTOPLASMIC PROTEIN BINDING TO TNFα mRNA AU‐RICH INSTABILITY ELEMENT IN HUMAN LYMPHOID CELLS
Authors: Henics Tamás
Published: October 2002

Cytoplasmic proteins with binding capability to AU‐rich instability determinant sequences (ARE) of tumour necrosis factor α (TNFα) mRNA 3′ untranslated region (3′UTR) were assessed in human lymphoid cells.In vitro label transfer experiments using wild type as well as mutant sequences in which the 70 nucleotide‐long AUUUA pentamer‐containing portion of the 3′UTR had been deleted conferred binding specificity to five major activities of 22/25‐, 38/40‐, 50‐, 60‐ and 80‐kDa proteins in cytoplasmic extracts of peripheral blood mononuclear cells (PBMCs). Cytochalasin‐induced disarrangement of the F‐actin‐based microfilament system led to a Triton X‐100‐insoluble to soluble redistribution of these binding activities. No such changes were observed in Jurkat tumour cells. Combination of in vivo UV‐crosslinking and in vitro label transfer experiments revealed considerable differences in RNA association between proteins of the same cell type as well as between proteins of identical molecular weight (Mw) derived from either PBMCs or Jurkat cells. Our findings may explain some aspects of differential regulation of interleukin 2 (IL‐2) and TNFα mRNA stability upon microfilament disruption in human PBMCs observed in an earlier study. These results also suggest that the physical state of cytoplasmic structural environment might contribute to important regulatory processes regarding key elements of eukaryotic mRNA metabolism, such as modulation of stability. Finally, these data highlight the possibility that the often observed disorganization of the cytoskeleton in tumour cells may partly be responsible for the maintenance of the neoplastic state, a phenomenon that potentially involves ARE‐AUBP interactions.
PEROXYNITRITE ACTIVATES K+‐Cl−COTRANSPORT IN HUMAN ERYTHROCYTES
Authors: Grzelak Agnieszka , Mazur Janusz , Bartosz Grzegorz
Published: October 2002

Peroxynitrite was found to induce the release of K+ via the Na+/Cl− cotransport system, as do other oxidants. Since peroxynitrite is formed in vivo, its presence could contribute to a pathological dehydration of red blood cells.
EARLY PROGRESSION FROM DIMETHYL SULFOXIDE‐INDUCED G0/G1 ARREST IN L1210 CELLS
Authors: Kudo Hideki , Nakayama Tohru , Mano Yoshihiro , Suzuki Satoe , Sassa Shuji , Sakamoto Shinobu
Published: October 2002

Recently, dimethyl sulfoxide (DMSO) has been used as a convenient cryoprotectant for stem cells in stem cell transplantation using allogenic peripheral blood or umbilical cord blood. As the stem cells have a multipotency, clarification of the extent of cell proliferation after transplantation is difficult. In the present study, DMSO gradually induced G0/G1 arrest in mouse leukemia L1210 cells with good cell viability. After removal of DMSO, the cells proliferated appropriately, resulting in expression of the DNA‐synthesizing enzymes thymidylate synthase and thymidine kinase within 6h, and the cells entering into S phase within 12h. The sequence was followed by the marked activation of both enzymes within 24h and the increase of bromodeoxyuridine (BrdU) immunoreactive (S phase) cells with rapid cell proliferation within 36h. In conclusion, mouse leukemia L1210 cells, which were treated with 1.5% DMSO for 96h, tolerated the treatment and reversed the cell cycle arrest within 36h.
ULTRASENSITIVITY IN (SUPRA)MOLECULARLY ORGANIZED AND CROWDED ENVIRONMENTS
Authors: Aon Miguel A. , Gomez‐Casati Diego F. , Iglesias Alberto A. , Cortassa Sonia
Published: October 2002

The ultrasensitive response of biological systems is a more sensitive one than that expected from the classical hyperbola of Michaelis‐Menten kinetics, and whose physiological relevance depends upon the range of variation of substrate or effector for which ultrasensitivity is observed. Triggering and modulation of the ultrasensitive response in enzymatic and cellular systems are reviewed. Several demonstrations of ultrasensitive behavior in cellular systems and its impact on the amplification properties in signalling cascades and metabolic pathways are also highlighted. It is shown that ubiquitous cytoskeletal proteins may up‐ or downmodulate ultrasensitivity under physico‐chemical conditions resembling those predominant in cells.