bluuebunny/crossref_metadata_2025 huggingface dataset:
INDUCTION OF CELLULAR PROCESSES CONTAINING COLLAGENASE AND RETINOID BY INTEGRIN‐BINDING TO INTERSTITIAL COLLAGEN IN HEPATIC STELLATE CELL CULTURE
Authors: SATO MITSURU , KOJIMA NAOSUKE , MIURA MITSUTAKA , IMAI KATSUYUKI , SENOO HARUKI
Published: September 2002

Cultered hepatic stellate cells were induced to elongate long, multipolar cellular processes by interstitial collagen gel used as a substratum, as compared to flattened or round cell shapes on polystyrene surface or on Matrigel containing the basement membrane components, respectively. The process induction was inhibited by several reagents as follows: (1) anti‐integrin α2 antibody; (2) an oligopeptide, DGEA, an integrin‐binding sequence in type I collagen molecule; (3) wortmannin, a phosphatidylinositol 3‐kinase inhibitor. Protein tyrosine phosphorylation was enhanced throughout cells including cellular processes by culturing on type I collagen gel. Dual fluorescence staining showed that the core of the processes contained microtubules, whereas the periphery of the processes comprised fibrillar actin. Thus, the process extension was found to depend on integrin‐binding to type I collagen fibres, followed by signal transduction and cytoskeleton assembly. The cellular processes included interstitial collagenase and vitamin A‐containing lipid droplets. The lipid droplets and vitamin A‐autofluorescence were increased by retinyl acetate addition to the culture medium, suggesting an important role of processes in hepatic stellate cell function.
PROTEINS INVOLVED IN MEMBRANE TRANSPORT BETWEEN THE ER AND THE GOLGI APPARATUS: 21 PUTATIVE PLANT HOMOLOGUES REVEALED BY DBEST SEARCHING
Authors: ANDREEVA A. V. , KUTUZOV M. A. , EVANS D. E. , HAWES C. R.
Published: September 2002

Numerous proteins have been identified in yeast and mammalian cells which are involved in trafficking between the endoplasmic reticulum and the Golgi apparatus. A great number of partial cDNA sequences now available from the two major plant model species, Arabidopsis thaliana and Oryza sativa, makes it possible to identify putative plant homologues of known genes/proteins from non‐plant species. The authors used this approach to screen the database of Expressed Sequence Tags (dbEST) in order to detect plant homologues of proteins involved in membrane transport between ER and Golgi. Availability of these partial sequences will facilitate the screening of cDNA and genomic libraries otherwise performed using heterologous probes derived from animal and yeast genes. As the plant Golgi complex differs in many respects from its mammalian and yeast counterparts, the dbEST clones found can be directly used for various functional assays (immunoprecipitation, two‐hybrid analysis, transgenic plants etc.) to test the exact roles of the encoded proteins and identify their functional partners, some of which may be specific for plants.
CREATINE KINASE AND CREATINE KINASE ISOENZYME RESPONSES TO HEAT STRESS
Authors: TERBLANCHE S. E. , NEL W.
Published: September 2002

During this investigation the effects of heat acclimation and exercise on creatine kinase and creatine kinase BB isoenzyme responses in various tissues and serum of male Sprague—Dawley rats were ascertained. Forty rats were randomly divided into two groups of 20 rats each. One group was housed at 22±1°C and the other at 33±1°C. Each of the two groups were subdivided into two subgroups of ten rats each. One subgroup of each group was subjected to a programme of treadmill running of progressive intensity over a period of 6 weeks at the temperature at which it was housed while the other served as a resting control. At the end of the acclimation programme the rats were running at 23m/min for 80min. On the day of sacrifice all four subgroups were subjected to a discontinuous exercise protocol (10min running alternated by a 2‐min rest period; repeated three times) at 30±1°C on a rodent treadmill at 23m/min. The tissues investigated were kidney, heart and muscle. The rats were anaesthetized with pentobarbital sodium (6mg/100g body mass) injected intraperitoneally. The tissues were freeze‐clamped and stored in liquid air until analysed. The body temperature of the four subgroups at the end of the experimental protocol were not significantly different. Acclimation at 33±1°C resulted in significantly lower creatine kinase activity levels. Exercise at 30±1°C also resulted in decreased creatine kinase activity levels in both acclimated groups. A similar trend was observed regarding creatine kinase BB isoenzyme activity levels, especially in kidney.
EFFECTS OF INTERLEUKIN‐6 ON PROLIFERATION AND PROTEOGLYCAN METABOLISM IN ARTICULAR CHONDROCYTE CULTURES
Authors: Jikko Akitoshi , Wakisaka Takashi , Iwamoto Masahiro , Hiranuma Hiroko , Kato Yukio , Maeda Takashi , Fujishita Masami , Fuchihata Hajime
Published: September 2002

Interleukin‐6 (IL‐6) levels are markedly increased in the synovial fluid of patients with rheumatoid arthritis or osteoarthritis. However, the effects of IL‐6 on proliferation and proteoglycan metabolism in articular cartilage are not known. We demonstrated here the effects of human recombinant (hr) IL‐6 on proliferation and proteoglycan metabolism in rabbit articular chondrocyte cultures. In vitro, these cells proliferated and produced abundant extracellular matrices. We found that 1–10ng/ml of hrIL‐6 inhibited proliferation to approximately 65% of control levels and suppressed colony formation induced by bFGF in soft agarose. The same concentration of hrIL‐6 depressed proteoglycan synthesis to approximately 60% of control levels. Moreover, hrIL‐6 significantly enhanced proteoglycan degradation induced by hrIL‐1β, although hrIL‐6 alone did not affect proteoglycan degradation. These findings suggest that IL‐6 is a negative regulator for chondrocyte proliferation and articular cartilage metabolism.
TERMINAL DIFFERENTIATION AND LEFT‐HANDED Z‐DNA: A REVIEW
Authors: Gagna Claude E. , Kuo Hon‐reen , Lambert W.C
Published: September 2002

Nucleic acids control the expression of genes, and different conformations of DNA structure may regulate cell death. Left‐handed Z‐DNA, which is speculated to function as a transcriptional enhancer, may be directly influenced by the destructive effects of terminal differentiation. The nicking‐denaturation of double‐stranded Z‐DNA could possibly initiate and enhance terminal differentiation within specific tissues.
CONNEXIN EXPRESSION IN HUNTINGTON'S DISEASED HUMAN BRAIN
Authors: Vis J. C. , Nicholson L. F. B. , Faull R. L. M. , Evans W. H. , Severs N. J. , Green C. R.
Published: September 2002

In Huntington's diseased human brain, it is in the caudate nucleus (CN) and globus pallidus (GP) of the basal ganglia where nerve cell death is seen most dramatically. The distribution of five gap junction proteins (connexins 26, 32, 40, 43 and 50) has been examined in these areas in normal and Huntington's diseased human brain using immunohistochemical techniques. There was no Cx50 expression observed and Cx40 was localized in the endothelial cells of blood vessels, with the Huntington's diseased brains having more numerous and smaller blood vessels than normal tissue. Cx26 and Cx32 revealed a similar distribution pattern to each other in both normal and diseased brains with little labelling in the CN but clear labelling in the GP. Cx43, expressed by astrocytes, was the most abundant connexin type of those studied. In both normal and diseased brains Cx43 in the GP was homogeneously distributed in the neuropil. In the CN, however, Cx43 density was both increased with Huntington's disease and became located in patches. Glial fibrillary acidic protein(GFAP) staining of astrocytes was also highly increased in the CN compared with normal brains. These labelling patterns indicate a reactive astrocytosis around degenerating neurons with an increased expression of astrocytic gap junctions. The enhanced coupling state between astrocytes, assuming the junctions are functional, could provide an increased spatial buffering capacity by the astrocytes in an attempt to maintain a proper environment for the neurons, helping promote neuronal survival in this neurodegenerative disorder.
INTERACTION BETWEEN PROTEIN KINASE C AND SPHINGOMYELIN/CHOLESTEROL
Authors: Jiang Yan , Pan Zui , Chen Jian Wen
Published: September 2002

Physical characteristics of binding of protein kinase C with sphingomyelin/cholesterol lipid bilayers were analysed using three complementary approaches: acrylodan fluorescence, fluorescence energy transfer and quenching of tryptophan fluorescence. It was demonstrated that sphingomyelin/cholesterol lipid membranes were available for protein kinase C binding. The intensity of the binding was dependent on the sphingomyelin content. The results of quenching of intrinsic tryptophan fluorescence showed that the enzyme molecule penetrated the sphingomyelin/cholesterol lipid bilayer to the C‐16 position of labeled fatty acid probes. Our results also showed sphingomyelin itself restrains protein kinase C activity. A possible explanation for our results is that caveolae function as signaling storage devices.
TRANSCRIPTIONAL ACTIVATION OF HEME OXYGENASE−1 GENE IN MOUSE SPLEEN, LIVER AND KIDNEY CELLS AFTER TREATMENT WITH LIPOPOLYSACCHARIDE OR HEMOGLOBIN
Authors: Oshiro Satoru , Takeuchi Hiroki , Matsumoto Midori , Kurata Shun‐Ichi
Published: September 2002

Heme oxygenase (HO)−1 catalyzes the conversion of heme to biliverdin, iron and carbon monoxide. HO−1 is induced by many reagents including heme, Hb and lipopolysaccharide (LPS). LPS is known to activate the HO−1 gene in cultured mouse liver and macrophage cells through oxidative activation of NF‐κB. But little is known about the effect of LPS and Hb on the HO−1 gene in living organisms. To study this issue, we examined the HO−1 and its mRNA levels in mouse liver, spleen and kidney after intravenous administration of LPS and Hb. On LPS treatment, the amount of HO−1 and its mRNA increased markedly mainly in mouse spleen, but on Hb treatment the amounts of HO‐1 and its mRNA increased slightly only in liver. Run‐off transcription assay supported the above results and band shift assays also revealed that LPS significantly activates an NF‐κB‐like factor in spleen cells, while Hb slightly activates it in liver cells. According to our previous study, a small amount of Hb injected to mouse is selectively taken up by liver as Hb—haptoglobin complex.

These results suggest different pathways for the HO gene activation in mouse organs; one by LPS in spleen cells and the other by Hb in liver cells.
EFFECTS OF 1α,25‐DIHYDROXYCHOLECALCIFEROL AND CORTISOL ON THE GROWTH AND DIFFERENTIATION OF PRIMARY CULTURES OF MOUSE MAMMARY EPITHELIAL CELLS IN COLLAGEN GEL
Authors: Kanazawa Takuya , Enami Jumpei , Kohmoto Kaoru
Published: September 2002

We examined the effects of 1α,25‐dihydroxycholecalciferol (1,25‐DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10−11–10−9m) of 1,25‐DHCC stimulated growth of the cells in a collagen gel matrix culture in serum‐free DMEM+Ham's F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8‐fold the control after 6d in culture. In contrast, supraphysiological concentrations (10−8–10−7m) of 1,25‐DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose‐dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25‐DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25‐DHCC also affected casein synthesis by cells cultured in a serum‐free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10−11–10−9m) and inhibiting it above 10−8m. In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25‐DHCC. These results suggest a physiological role for 1,25‐DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25‐DHCC does not substitute for glucocorticoids in the differentiation of the cells.
CELL PAIRING AND METHYLATION IN TETRAHYMENA THERMOPHILA ARE ALTERED BY EXOGENOUS HOMOCYSTEINE
Authors: Wolfe Jason , Sasson Shmuel Ben , Ron Arie
Published: September 2002

Homocysteine is causally associated with birth defects such as spina bifida, and with premature vascular disease. We have investigated the effects of homocysteine on a cell—cell interaction in a fundamental eukaryotic system, the free‐living ciliate Tetrahymena. Exogenously added homocysteine inhibits cell pairing in a dose‐dependent manner. These effects are exacerbated by adenosine, which by itself has little demonstrable influence on pairing. S ‐adenosylhomocysteine (SAH) is a product of the reaction between adenosine and homocysteine, and is an inhibitor of methyl transferases. We therefore predicted that protein methylation would be significantly inhibited by homocysteine. A direct test of that hypothesis involved a demonstration that incorporation of an isotopically labeled methyl group from methionine into proteins was significantly reduced by homocysteine. The undermethylated proteins are of low molecular weight, and might correspond to known methylatable signaling proteins. We show that vanadate, an inhibitor of protein phosphatase, also inhibits cell pairing, and that the effects of vanadate and homocysteine are additive. This is the first demonstration that methylation and possibly phosphorylation play a regulatory role in cell—cell interactions in ciliates.
DNA‐END BINDING ACTIVITY OF KU IN SYNCHRONIZED CELLS
Authors: Chou Li‐Fen , Chou Wen‐Gang
Published: September 2002

Three different types of cells were synchronized by various methods and DNA‐end binding (DEB) activities of Ku were compared with asynchronous controls. In CHO K1 cells synchronized in G1 phase by serum starvation and in S phase by serum refeeding, DEB activity was reduced in S cells but remained unchanged in G1 cells. However, the same type of cells synchronized in G1/S phase by double thymidine block and in S phase by releasing the blockage, have the same DEB activity as asynchronous controls. A similar result was found in RKO and HeLa cells synchronized by the latter method. Arresting cells in mitosis with nocodazole also generated different cell cycle effects. Ku activity was reduced in CHO K1 and RKO cells, but not in HeLa cells after treatment with nocodazole. In phase‐enriched cells separated by centrifugal elutriation, DEB activities were similar at different stages of the cell cycle in all three types of cells. Thus, different synchronization procedures can give very different values of Ku activity in a cell type‐dependent manner. Results from elutriated cells are consistent, and suggest DEB activity of Ku does not change with the cell cycle.
INTERLEUKIN‐6‐INDUCED PRODUCTION OF TYPE II ACUTE PHASE PROTEINS AND EXPRESSION OF junB GENE ARE DOWNREGULATED BY HUMAN RECOMBINANT GROWTH HORMONE IN VITRO
Authors: Derfalvi Beata , Igaz Peter , Fulop Kristof A. , Szalai Csaba , Falus Andras
Published: September 2002

Growth hormone (GH), given therapeutically in many human diseases, is able to modulate the maturation and function of many cells of immune system. The present study demonstrates the effect of human recombinant GH on the production of acute phase proteins (APP) as well as on the gene expression of junB proto‐oncogene on human hepatoma cell line, HepG2. When applied alone GH resulted in an increase in the transcription of junB proto‐oncogene within 30min. The production of α2‐macroglobulin, haptoglobin and fibrinogen was also enhanced by rhGH treatment. However, both IL‐6‐stimulatedjunB gene expression (junB mRNA) and biosynthesis of type II APP (α2‐macroglobulin, fibrinogen, haptoglobin) were strongly inhibited by the GH. The results indicate that GH has a modulatory role in regulating inflammation both in the absence and presence of IL‐6. These findings call for further in vivo studies to determine the potential anti‐inflammatory actions of GH therapy.
ARE CELLS RESCUED FROM ‘LOW DENSITY DEATH’ BY CO‐OPERATION BETWEEN PHOSPHOLIPASES C AND D?
Authors: Rasmussen Morten , Rasmussen Leif
Published: September 2002

Cells of the ciliate Tetrahymena thermophila die when transferred at low density to a lipid‐free nutritionally complete medium. This death is prevented and they will start to proliferate if protein kinase C is activated and this activation is sustained. We propose that this takes place in two stages. Firstly, the phospholipase C pathway beginning with and specific for phosphatidylinositol leads to the formation of diacylglycerol and inositol tris ‐phosphate. Diacylglycerol activates protein kinase C, and inositol tris ‐phosphate via Ca2+phospholipase D (PLD). Secondly, the protein kinase C response can now be sustained by diacylglycerol produced by phospholipase D, using phosphatidylcholine and phosphatidylserine as substrates. Should this switching from PI‐specific phospholipase C (PLC) to phospholipase D fail, then the cell will die in the course of milliseconds during the minutes following inoculation.
EFFECT OF THE SERINE PROTEASE INHIBITOR N‐TOSYL‐L‐PHENYLALANINE‐CHLOROMETHYL KETONE (TPCK) ON MCF‐7 MAMMARY TUMOUR CELLS GROWTH AND DIFFERENTIATION
Authors: Horman Sandrine , Del Bino Giacinta , Fokan Dominique , Mosselmans Roger , Galand Paul
Published: September 2002

Previous studies from this and other laboratories indicated that the oestrogen‐regulated heat shock protein HSP27 is involved in the control of MCF‐7 cells growth and differentiation, as it also appears to be in other cell types, including osteoblasts and HL‐60 cells. In the latter instance, induction of differentiation is associated with the downregulation of myeloblastin, a serine protease now identified as proteinase 3 (hence its designation as PR3/Mbn), mirrored by an increase in the cellular content of the small heat shock protein HSP27, a substrate to this enzyme. Besides, antisense inhibition of PR3/Mbn production sufficed for inducing HL‐60 cells monocytic differentiation. This prompted us to examine the hypothesis that a post‐translational control on HSP27 levels (and by this on differentiation) by a serine protease might also be operating in human mammary tumour cells. As part of our attempt to evaluate this hypothesis, the present work consisted of testing the effects of a treatment of MCF‐7 cells with the serine protease inhibitor N‐tosyl‐L‐phenylalanine‐chloromethyl ketone (TPCK). Our data show that this resulted in a four‐fold increase in HSP27 content, associated with a 2.5‐fold decrease in growth rate, the formation of cytoplasmic vesicles and increased secretion of 52kDa peptides, identified by Western immunoblot as the isoforms of the oestrogen‐regulated protein, cathepsin D. TPCK only affected growth in MDAMB‐231 cells (in which HSP27 levels are very low and remained below MCF‐7 cells basal levels after treatment) and failed to affect L929 cells, in which the hsp27 gene is silent. This provides circumstantial support for the assumption that effects of TPCK on the MCF‐7 cells phenotype are linked to the associated increase in HSP27 content. Our recent demonstration that MCF‐7 cells do in fact express PR3/Mbn fits with our concept and opens the way to test it directly, using antisense strategy.
INDUCTION OF DNA SYNTHESIS OR APOPTOSIS IN MAMMALIAN NUCLEI BY XENOPUS EGG EXTRACTS THAT FAIL TO SUPPORT THE REPLICATION OF SPERM CHROMATIN
Authors: Logothetou‐Rella Helen , Sun Wei‐Hsin , Brooks Robert F.
Published: September 2002

Cell‐free extracts of Xenopus eggs are widely used to study DNA replication in higher eukaryotes. However, with Xenopus sperm chromatin as template, it is a common experience that the efficiency of replication varies from extract to extract, for reasons that are unclear. Here we show that the majority of extracts unable to support sperm replication are nevertheless able to induce DNA synthesis in intact mammalian nuclei. Those that do not, induce apoptosis.
DIFFERENCES IN THE MICROTUBULE ORGANIZATION BETWEEN NORMAL AND CML GRANULOCYTES AFTER STIMULATION WITH CHEMOTACTIC PEPTIDE
Authors: Naik Nishigandha , Rigaut Jean‐Paul , Advani Suresh , Bhisey Avinash
Published: September 2002

Chemotaxis of polymorphonuclear leukocytes (PMNL) from chronic myeloid leukemia (CML) patients followed in a gradient of a chemotactic peptide n‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) is consistently defective in all the phases of the disease. Chemoattractant‐induced polymerization of cytoskeletal proteins (actin and tubulin) plays a major role in regulation of cell shape and cellular motility. To study the role of microtubules in defective chemotaxis, we have compared fMLP‐induced alterations in organization of microtubules in PMNL from CML patients with those from normal subjects by laser confocal microscopy. Our analysis shows differences in microtubule organization between normal and CML PMNL and suggests that both nucleation of new microtubule and elongation of pre‐existing microtubules are essential for PMNL chemotaxis.
GLUCOCORTICOIDS ENHANCE SERUM DEPRIVATION‐ BUT NOT CALCIUM‐INDUCED CYTOTOXICITY IN RAT C6 GLIOMA CELLS
Authors: Morita Kyoji , Wong Dona L.
Published: September 2002

While glucocorticoids have been shown to exacerbate calcium‐induced neuronal damage, little is known about the effects of these hormones on calcium‐induced damage to glial cells. Here we examine the effect of synthetic glucocorticoid dexamethasone on calcium ionophore A23187 and serum deprivation‐induced damage to rat C6 glioma cells. Treatment of the glioma cells with A23187 reduced cell viability, similar in extent to that observed with serum deprivation. Both A23187 and serum deprivation caused cell damage without degradation of the genomic DNA into nucleosomic fragments. In addition, the reduction in cell viability caused by A23187 was not significantly altered by DEX at concentrations enhancing serum deprivation‐induced cell death. These results suggest that the cytotoxic effect of A23187 on glial cells may be mediated through a mechanism different from that underlying serum deprivation‐induced cell death, and that, in contrast to calcium‐induced neuronal damage, calcium‐induced damage to glial cells is likely to be insensitive to glucocorticoids.
PROTEOLYTIC ACTIVITY IN INTACT SHEETS OF POLARIZED EPITHELIAL CELLS AS DETERMINED BY A CELL‐PERMEABLE FLUOROGENIC SUBSTRATE
Authors: Karlsson Jan‐Olof , Lundquist Jenny , Skoglund Ida , Nilsson Mikael
Published: September 2002

The purpose of the present investigation was to develop a system for continuous evaluation of extralysosomal proteolytic activity and its regulation in polarized epithelial cells. Filter inserts containing a tight monolayer of primary cultured pig thyrocytes were placed in a thermostated aluminium block. The cell‐permeable, fluorogenic calpain and proteasome substrate succinyl‐Leu‐Leu‐Val‐Tyr‐7‐amino‐4‐methylcoumarin was added to the apical buffer and fluorescence changes were continuously measured via the fibre optics of a luminometer held at a fixed distance from the cell layer. Basal proteolytic activity was reduced by 60–70% by the proteasome inhibitor lactacystin. Proteolysis was increased within a few minutes after application of Ca2+‐mobilizing agents (ionomycin, 4‐bromo‐A23187, thapsigargin and maitotoxin). Forskolin and staurosporine also enhanced the proteolytic activity. We conclude that Ca2+mobilization, and possibly also changes of protein kinase activity, rapidly increase non‐lysosomal proteolysis in the intact thyroid epithelium.
CHANGE IN THE ACTIVITY OF LECTINS IN CELL STRUCTURE OF WINTER WHEAT CROWN UNDER THE ACTION OF LOW‐TEMPERATURE HARDENING AND FUSICOCCIN
Authors: Komarova E. N. , Trunova T. I. , Vyskrebentseva E. I.
Published: September 2002

Haemagglutinating activity was determined in cell walls and total cell organelles of crown cells of Winter wheat (Triticum aestivum L.) plants. The effect of fusicoccin (FC) was investigated using fractions obtained from plants hardened for 7 days at 2°C and from untreated plants. FC concentration (5×10−7m) increased the frost resistance of the plants. The temporal pattern of lectin activity during hardening could be described by a single‐peak curve. In the cell wall fraction, the highest activity manifested itself after one‐day hardening, and in the fraction of organelles it peaked after five‐days hardening. The carbohydrate specificity of lectins also changed during hardening; cell wall lectins completely lost their capacity for interaction with uridine diphosphoglucose, glucose 6‐phosphate, D‐galactosamine, and N‐acetylglucosamine and the lectins of organelles retained some affinity only for amino sugars. After hardening the test plants, the activity of the lectins increased substantially in the cell walls and plastids, decreased in the nuclei, and was practically flat in mitochondria and microsomes. Consequently, low temperature and FC with their antistress effect improved frost resistance and stimulated the activity of the lectins of some cell structures of the tillering node of winter wheat. A similar action of low temperature and FC in increasing the activity of lectins of plastids was found. Further information was obtained on the subcellular localization of lectins providing additional information on their possible participation in the development of frost resistance of winter wheat.
INFLUENCE OF CYTOSKELETAL AGENTS ON THE RESPIRATORY ELECTRON TRANSPORT PATHWAYS IN THE CELLS OF WINTER WHEAT LEAVES
Authors: Abdrakhimova Y. R. , Khokhlova L. P. , Abdrakhmanova A. F. , Abdrakhimov F. A.
Published: September 2002

The effects of actin and tubulin polymerization inhibitors on the respiratory electron transport pathway activities were investigated using abscisic acid (ABA)‐ and cold‐treated winter wheat seedling leaves. In unstressed control plants, cytochalasin B (15μm) decreased the capacity of the cytochrome pathway, but stimulated the cyanide‐resistant pathway, whereas oryzalin (15μm) produced the opposite effects. Cold hardening (3°C for 7 days) and ABA treatment 30μm changed the respiratory pattern in a similar manner to cytochalasin B but to lesser effects. This points to cold‐ and ABA‐induced reduction in microfilament sensitivity to these drugs and hence stabilization of actin‐dependent processes. In contrast, oryzalin had only weak effects on control samples and its effects were strengthened in the presence of the cytoskeleton‐modifying factors. The data suggest that the potential targets for the agent either increase and/or the degree of involvement of microtubules in the respiratory chain regulation, and therefore that the cytoskeleton can modify the functioning of the respiratory electron transport pathways in winter wheat cells.
LOW IN VIVO LEVELS OF HUMAN ANX7 (ANNEXIN VII) GENE EXPRESSION ARE DUE TO ENDOGENOUS INHIBITORY PROMOTER SEQUENCES
Authors: Srivastava Meera , Pollard Harvey B.
Published: September 2002

ANX7 is a Ca2+/GTP‐dependent membrane fusion protein, which is involved in regulating exocytotic secretion. While the nullizygous ANX7 (−/−) knockout mutation in the mouse is lethal, ANX7 is nonetheless the least expressed member of the annexin gene family in mammalian and lower organisms. To investigate the basis for low ANX7 expression level we have studied the human ANX7 promoter. In our early cloning studies we learned that the human ANX7 promoter lacks TATA and CCAAT boxes, but is GC rich and contains several SP1 binding sites. To study how expression of the ANX7 gene is controlled, we fused nested deletion mutants of the 5′‐flanking region to a luciferase reporter gene. We find that the maximal level of luciferase activity is obtained by a construct comprising the domain −889/+34. However, a repressor effect observed in the region between −240 and +34 is dramatically eliminated either by the inclusion of 177bp upstream sequences, or by the presence of an upstream SV40 enhancer sequences. ANX7 gene expression is also cell type specific and is subject to different transcriptional controls in different cellular environments. The fact that the SV40 enhancer sequences not only eliminates the suppression of luciferase activity in the inhibitory region of the ANX7 promoter, but also reverses the inhibitory effect of phorbol ester, suggests that endogenous cis‐acting elements or transacting factors may be responsible for the physiologically low level ofANX7 protein expression.
A NUCLEAR MATRIX PROTEIN RELATED TO INTERMEDIATE FILAMENTS PROTEINS IS A MEMBER OF THE COMPLEX BINDING ALPHOID DNAIN VITRO
Authors: Enukashvily N. I. , Lobov I. B. , Kukalev A. , Podgornaya O. I.
Published: September 2002

A complex of three proteins (of 80, 70, 58kDa—p80, p70, and p58, respectively) with the ability to bind alphoid DNA (α‐satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the α‐satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA‐binding proteins, p70 and p80, and a non‐DNA‐binding one, p58, which enhances the specificity of binding to the α‐satDNA. GMSA, SDS‐PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP‐A, CENP‐B, CENP‐C, CENP‐G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF‐related protein that is able to bind specifically to α‐satDNA in vitro and that this protein is distinct from the lamins.
TRANSLOCATION OF P53‐REGULATED LAMININ RECEPTORS IN PRO‐APOPTOTIC MICROCIRCUMSTANCE OF HUMAN VASCULOGENESIS INHIBITION
Authors: Wang Lin , Beihua Kong , Minxing Duan , Youzhong Zhang , Shaoling Li , Chang Zengyi
Published: September 2002

It is not clear what type of cells will inhibit vasculogenesis and under what circumstances. Here we examined the human spontaneous abortion villi and discovered that cells in the vasculogenesis‐inhibited environment appear to be pro‐apoptotic as indicated by strong staining of bak. In particular, an increasing laminin receptor (LNR) expression in cytoplasm and on apical membrane, being most likely mediated by p53 because of coexpression, was observed in the pro‐apoptotic syncytiotrophoblasts. The transcriptional increase and translocation of the receptors to the cytoplasm and apical membrane may be used as a pathological marker, indicating a change of the structure and function of integrins in the non‐vasculogenesis micro‐environment.
CENTROSOME ALTERATIONS INDUCED BY FORMAMIDE CAUSE ABNORMAL SPINDLE POLE FORMATIONS
Authors: Schatten Heide , Hueser Christopher N. , Chakrabarti Amitabha
Published: September 2002

The formation of the bipolar mitotic apparatus depends on accurate centrosome organization which is crucial for the separation of the genome during cell division. While it has been shown that mutations and overexpression of centrosome proteins (Brinkley and Goepfert, 1998; Pihan et al., 1998) can cause abnormal spindle pole formation, here we report that damages to centrosome structure caused by the chaotropic agent formamide will cause multipolar mitoses upon recovery from the effect when applied at first cell division in sea urchin eggs. Formamide was used as a chemical tool to manipulate centrosome structure and to investigate the effects on microtubule organization. When 1–1.5m formamide was administered for 30min at prometaphase of first cell division, microtubules were disassembled and centrosomes compacted into dense spheres around highly condensed chromatin. Upon recovery from formamide, centrosomes decompacted and attempted to form various mitotic organizations. Normal recovery (and attempts of recovery) to bipolarity was possible in five percent of cells treated with 1–1.5m formamide for 30min, but abnormal patterns of spindle formation were observed in all other cells, which included mono‐ (20%), tri (45%), and multipolar (30%) formations organized by mono‐, tri‐, and multipolar centrosome clusters. When cells were treated with 1.5m formamide for 90min, centrosomes became pulverized and fragmented and only monopolar mitotic formations were observed upon recovery. These results are highly reproducible and reveal that abnormalities in centrosome structure can lead to abnormal mitosis which is not caused by mutation or overexpression of centrosome proteins.
EXTENDED CHROMATIN FIBRES: CRYSTALLINITY, MOLECULAR ORDER AND REACTIVITY TO CONCANAVALIN‐A
Authors: De Vidal Campos Benedicto
Published: September 2002

The present study was undertaken to test the reproducible formation of the extended chromatin fibres (ECF), beads and superbeads, and detect molecular order and crystallinity by optical anisotropies in those structures. Chicken erythrocyte smears and mouse liver cell imprints were treated with 2.0–3.0 m NaCl solution in 1% Triton‐X100 vertically prior to staining with 0.025% toluidine blue at pH4. Detection of birefringence and colours of abnormal dispersion of birefringence (ADB) following toluidine blue binding to DNA revealed that the DNA molecular order and crystallinity in decondensed and condensed chromatin are preserved after ECF formation. The tests for Con‐A binding to mannose/glucose residues of glycoproteins was confirmed within nuclei, and in the ECF, beads and superbeads. ECF formation was not regular. Clumped cells did not show ECF, although chromatin mobility was observed within the nuclei. Electron microscopy demonstrated that after treatment of nuclei with 0.77 m NaCl ECF always spread from the nuclei, in clumped nuclei the fibres aggregated instead of spreading.
RELEASE OF MITOTIC DESCENDANTS BY GIANT CELLS FROM IRRADIATED BURKITT'S LYMPHOMA CELL LINES
Authors: Erenpreisa Jekaterina A. , Cragg Mark S. , Fringes Birgitta , Sharakhov Igor , Illidge Timothy M.
Published: September 2002

Polyploid giant cells are produced as part of the response of p53 mutant Burkitt's lymphoma cell lines to high doses of irradiation. Polyploid giant cells arise by endo‐reduplication in the first week after a single 10 Gray dose of irradiation. Within the giant cells a sub‐nuclear structure is apparent and within this, sub‐nuclear autonomy is evident, as displayed by independent nuclear structure and DNA replication in different parts of the nucleus. The majority of these cells soon die as apoptotic polykaryons. However, approximately 10–20% of giant cells remain viable into the second week after irradiation and begin vigorous extrusion of large degraded chromatin masses. During the second week, the giant cells begin to reconstruct their nuclei into polyploid ‘bouquets’, where chromosome double‐loops are formed. Subsequently, the bouquets return to an interphase state and separate into several secondary nuclei. The individual sub‐nuclei then resume DNA synthesis with mitotic divisions and sequester cytoplasmic territories around themselves, giving rise to the secondary cells, which continue mitotic propagation. This process of giant cell formation, reorganization and breakdown appears to provide an additional mechanism for repairing double‐strand DNA breaks within tumour cells.
AUTOMATED DETECTION OF MORPHOLOGICAL ALTERATIONS DURING APOPTOSIS
Authors: Lertworasirikul Thawatchai , Bunyaratvej Ahnond , Kubota Fumio
Published: September 2002

A convenient means of measuring apoptosis is described using an automated analyzer (MF‐1; Sysmex), which normally provides rapid measurement of malarial parasites. By means of this MF‐1 procedure, apoptotic cells exhibited characteristic changes of light scatter (size) and fluorescence (DNA content) relating to cell shrinkage and nuclear fragmentation of apoptosis.
ROLE OF PHOSPHORYLATION OF THE CYTOPLASMIC DOMAIN OF THE α2‐MACROGLOBULIN RECEPTOR (LRP)
Authors: Djordjevic Julianne T. , Waterkeyn Jacqui G. , Hennessy Karen L. , Stanley Keith K.
Published: September 2002

The low density lipoprotein receptor‐related protein (α2MR/LRP) is a cell surface receptor which is present on most cells and tissues. We show that the 85kDa subunit, containing the transmembrane region and cytoplasmic domain is phosphorylated in vivo. Comparison of the phosphorylation of the low density lipoprotein receptor (LDLR) with a chimeric receptor containing the cytoplasmic domain of the α2MR/LRP (LDLR/LRP) showed that phosphorylation is exclusive to the cytoplasmic domain. Staurosporine, a general kinase inhibitor, resulted in a 40% lowering of phosphorylation of LDLR/LRP, but did not give rise to measurable changes in its membrane traffic in MDCK cells. The role of phosphorylation on degradation of the receptor was studied using inhibitors of lysosomal and proteasomal degradation. These studies showed that LDLR/LRP was rapidly turned over by proteasomal degradation but that this turnover was also not a consequence of phosphorylation.
RECOMBINANT HUMAN INTERLEUKIN‐8, BUT NOT HUMAN INTERLEUKIN‐1β, INDUCES BOVINE NEUTROPHIL MIGRATION IN AN IN VITRO CO‐CULTURE SYSTEM
Authors: Lee Jaiwei , Zhao Xin
Published: September 2002

A co‐culture system was established by culturing a bovine mammary epithelial cell line (MAC‐T) and a bovine aortic endothelial cell line on calf tail collagen pre‐coated inserts. This system allowed us to study bovine neutrophil migration across endothelium, extracellular matrix (ECM), and epithelium in the correct sequence and direction in vitro. The effect of recombinant interleukin‐1β (rHIL‐1β) and interleukin‐8 (rHIL‐8) on bovine neutrophil migration was investigated using this system. rHIL‐8 stimulated bovine neutrophil migration in a dose‐dependent fashion. The level of migrating bovine neutrophils increased up to approximately 25% when 100ng/ml of rHIL‐8 was used. On the other hand, rHIL‐1β at concentrations up to 100ng/ml did not directly induce bovine neutrophil migration. Furthermore, pre‐incubation with 5ng/ml of rHIL‐1β in the co‐culture system for 4 or 24h failed to have any effect. These results suggest that IL‐8 plays an important role in neutrophil migration into bovine mammary glands during mastitis.
TRANSIENT CHAOS IN INTRACELLULAR DYNAMICS?
Authors: Gilbert D. A. , Visser G. , Ferreira G. M. N. , Hammond K. D.
Published: September 2002

Periodogram analyses of the temporal variations of several cellular oscillations occasionally reveal the existence of short duration bands containing a wide range of frequencies. The possibility is considered that these are due to the transient compliance with chaotic conditions.
INVOLVEMENT OF PI 3‐KINASE, PKA AND PKC IN PDGF‐ AND TGF‐β‐MEDIATED PREVENTION OF 2‐DEOXY‐D‐RIBOSE‐INDUCED APOPTOSIS IN THE INSECT CELL LINE, IPLB‐LdFB
Authors: Ottaviani Enzo , Barbieri Daniela , Malagoli Davide , Kletsas Dimitris
Published: September 2002

Activation of phosphatidylinositol (PI) 3‐kinase, protein kinase A (PKA) and protein kinase C (PKC) is associated with the survival effect elicited by PDGF‐AB and TGF‐β1 against the apoptotic inducer 2‐deoxy‐D‐ribose (dRib) in the fat body cell line, IPLB‐LdFB, from the insect Lymantria dispar. dRib induces apoptosis and provokes mitochondrial membrane depolarization (MMD). The antioxidant N ‐acetyl‐L‐cysteine annuls only the first effect. These findings suggest that apoptosis and MMD are provoked by two different mechanisms, and that dRib induces apoptosis by oxidative stress.
EXPRESSION OF CELL ADHESION MOLECULE AND ALBUMIN GENES IN PRIMARY CULTURE OF RAT HEPATOCYTES
Authors: Hou De‐Xing , Arimura Miho , Fukuda Masahiro , Oka Tatsuzo , Fujii Makoto
Published: September 2002

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly‐ N‐p ‐vinylbenzyl‐D‐lactonamide (PVLA) coating of the dishes. In PVLA‐coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin α‐1 mRNA. The changes in mRNA levels of liver cadherin and integrin α‐1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA‐coated culture, hepatocytes expressed cadherin at higher levels to promote cell—cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.
INSIGHTS ON MEMBRANE FUSION
Authors: Jena Bhanu P.
Published: September 2002

Membrane fusion is a fundamental cellular process regulating intracellular transport, neurotransmission, enzyme secretion, hormone release, and the entry/exit of viruses, to name a few. Knowledge of how opposing bilayers fuse, besides advancing our understanding of these cellular processes, will provide us with the facts to ameliorate secretory defects and prevent cellular entry or exit of pathogenic viruses. In the last few years, great strides have been made in our understanding of the molecular machinery and mechanism of membrane fusion. In this Special Issue of Cell Biology International, entitled ‘Membrane fusion: machinery and mechanism’, we have tried to cover several aspects of this vital cellular process, providing insights on the machinery, mechanism and dynamics of the process. Membrane fusion studies reported in this Special Issue have been performed on whole cells, synaptic terminals, viruses, and fusion proteins.
FUNCTIONAL EXAMINATION OF MICROENCAPSULATED BIOENGINEERED INSULIN‐SECRETING BETA‐CELLS
Authors: Hamid M. , McCluskey J. T. , McClenaghan N. H. , Flatt P. R.
Published: September 2002

Clonal insulin‐secreting BRIN‐BD11 cells engineered by electrofusion were encapsulated inside natrium alginate beads and cultured in RPMI 1640 culture media. Acute insulin secretory responses to glucose and amino acids were compared between microencapsulated cells and non‐encapsulated cells maintained in monolayer culture. Encapsulated cells exhibited a 1.5‐fold, 2.9‐fold and 4.2‐fold increase (P< 0.001) in insulin release in response to 16.7mmol/l glucose, 10mmol/l L‐arginine and 10mmol/l L‐alanine respectively. Insulin output by non‐encapsulated cells was approximately 30% greater but the relative magnitudes of responses were similar. This is the first study to demonstrate the stability of cellular engineered insulin‐secreting cells encapsulated in alginate beads, illustrating the utility of this approach for cellular engineering and potential transplantation in diabetes.
INTEGRIN AND TALIN IN THE JELLYFISH PODOCORYNE CARNEA
Authors: Reber‐Müller Susanne , Studer Rolf , Müller Peter , Yanze Nathalie , Schmid Volker
Published: September 2002

We have isolated an integrin‐β and ‐α subunit from Podocoryne carnea (Cnidaria, Hydrozoa) and studied their expression in the life‐cycle and during cell migration, in vitro transdifferentiation and regeneration. Comparison of the integrin expression pattern with a Podocoryne talin homologue by RT‐PCR demonstrates that all three genes are maternal messages and continuously expressed in the life‐cycle, in medusa development and in all medusae tissues. In situ hybridisation experiments confirm co‐expression of both integrin subunits in the different life‐stages. Integrin expression was furthermore studied in isolated striated muscle induced to transdifferentiate to new cell types, or grafted on ECM where the muscle adheres and migrates. Integrin expression was maintained continuously throughout both processes. These results suggest that in Podocoryne carnea processes such as cell migration and differentiation are not controlled by up‐ or downregulation of alternative integrin subunits, but by a single integrin heterodimer which activates different downstream signalling cascades.
DIRECT NITRIC OXIDE IMAGING IN CULTURED HIPPOCAMPAL NEURONS WITH DIAMINOANTHRAQUINONE AND CONFOCAL MICROSCOPY
Authors: Chen Xiaochun , Sheng Chao , Zheng Xiaoxiang
Published: September 2002

A method for the direct measurement of intracellular nitric oxide (NO) production stimulated by penicillin G (PG) in cultured hippocampal neurons with diaminoanthraquinone (DAA) using laser scanning confocal microscopy (LSCM) was developed. Intracellular DAA fluorescence could specifically represent NO production based on two facts: (1) 3‐morpholinosydnonimine, a NO donor, could dose‐dependently increase DAA fluorescence; and (2) haemoglobin, a NO scavenger, could inhibit the increase of DAA fluorescence. The PG dose‐dependently increasd the intercellular level of glutamate (Glu, 5min after stimulation) and the intracellular NO production (30min throughout stimulation). The increase of NO production could be reversed by Nw‐nitro‐ l ‐arginine (a NO synthase inhibitor), and also by d (−)2‐amino‐5‐phosphonovaleric acid, a subtype of Glu receptor antagonist. These results revealed that DAA could be used to indicate real‐time and kinetic intracellular NO production of hippocampal neurons with higher sensitivity, specificity and accuracy.
TRANSCRIPTION OF Fcγ RECEPTORS IN DIFFERENT RAT LIVER CELLS
Authors: Løvdal Torunn , Berg Trond
Published: September 2002

Expression of Fcγ‐receptors (FcγRs) in different liver cells is poorly characterised. In the present study, transcription of FcγRs in different rat liver cells was examined by means of Northern blot analysis and reverse transcriptase‐polymerase chain reactions. We found that both Kupffer and liver endothelial cells produce mRNA for FcγRIIB2, FcγRIII and the γ‐chain, whereas the level of FcγRIIB1 mRNA is negligible. In contrast, parenchymal cells produce no FcγR mRNA.
PROTEIN KINASE C PATHWAY IS INVOLVED IN REGULATING THE SECRETION OF PROSTATIC ACID PHOSPHATASE IN HUMAN PROSTATE CANCER CELLS
Authors: Lin Ming‐Fong , Zhang Xiu‐Qing , Dean Jeanenne , Lin Fen‐Fen
Published: September 2002

The stimulated secretion of prostatic acid phosphatase (PAcP) has been known to be a hallmark of androgen action on human prostate epithelial cells for the last five decades. The molecular mechanism of androgen action on PAcP secretion, however, has remained mostly unknown. We investigated the molecular mechanism that promotes PAcP secretion in LNCaP human prostate carcinoma cells which express PAcP and are androgen‐responsive. Treatment with 12‐o‐tetradecanoyl phorbol‐13‐acetate (TPA), a protein kinase C (PKC) activator, resulted in an increased secretion of PAcP in a dose‐ and time‐dependent fashion. 4α‐Phorbol, a biologically inactive isomer of TPA, had no effect. This TPA stimulation of PAcP secretion was observed 2h after exposure, while TPA did not have a significant effect on the mRNA level even with a 6h treatment. A23187 calcium ionophore, known to mobilize cellular calcium which is a co‐factor of PKC, also activated PAcP secretion. This TPA stimulation of PAcP secretion was more potent than the conventional stimulating agent 5α‐dihydrotestosterone (DHT) at the same concentration of 50n m. Furthermore, the action of TPA and DHT on PAcP secretion was blocked by five different PKC inhibitors. Results also showed that DHT, as well as TPA, could rapidly modulate PKC activity. Therefore, PKC can regulate PAcP secretion, and may also be involved in DHT action on PAcP secretion.