bluuebunny/crossref_metadata_2025 huggingface dataset:
EFFECTS OF BETA MERCAPTOETHANOL ON THE PROLIFERATION AND DIFFERENTIATION OF HUMAN OSTEOPROGENITOR CELLS
Authors: INUI KENTARO , OREFFO RICHARD O.C , TRIFFITT JAMES T.
Published: September 2002

Antioxidants are known to influence metabolism and promote cell survival in a number of cell culture systems. However, their effects on the modulation of bone cell differentiationin vitroare not clearly defined. In the present studies we have investigated the effects of β‐mercaptoethanol (βME) and ascorbate alone and in combination on human osteoprogenitors derived from bone marrow fibroblasts. In primary marrow cultures, βME stimulated colony formation (2‐fold), alkaline phosphatase activity (3.5‐fold) and, increased DNA synthesis (8‐fold) after 21 days. Cell proliferation was increased significantly by βME during the first 4 days of a 10‐day culture period, indicating stimulation of marrow osteoprogenitor proliferation. Ascorbate did not significantly augment the effects of βME in primary cultures or long‐term cultures of passaged bone marrow fibroblasts. These findings indicate a potential beneficial role for βME addition for the optimal maintenance of colony formation, cell proliferation and differentiation of marrow osteoprogenitor cells in primary human bone marrow fibroblast cultures.
A NON‐INVASIVE METHOD FOR THE TIGHT ANCHORING OF CELLS FOR SCANNING FORCE MICROSCOPY
Authors: SCHILCHER K. , HINTERDORFER P. , GRUBER H. J. , SCHINDLER H.
Published: September 2002

Use of scanning force microscopy (SFM) for high resolution imaging of cell surfaces requires the cells to be tightly attached to substrates. Imaging of loosely adhered RBL‐2H3 cells enabled determination of the cell size and investigation of larger structures and pseudopodia but failed in resolving more detail. Immobilization under non‐invasive conditions via flexible crosslinkers containing a hydrophobic anchoring group enhanced resolution enormously. The cells were tightly attached to the substrates and were not removed by shear forces up to 80nN as determined in a flow through apparatus. Morphological structures and dynamic processes on cell surfaces were observed as well as structural changes after cell stimulation upon ionomycin treatment. Molecular or atomic resolution, however, was not attainable which is attributed to the displacement of the flexible cell surface due to shear forces arising from the scanning tip during contact mode.
RAPID SYNCYTIUM FORMATION BETWEEN HUMAN T‐CELL LEUKAEMIA VIRUS TYPE‐I (HTLV‐I)‐INFECTED T‐CELLS AND HUMAN NERVOUS SYSTEM CELLS: A POSSIBLE IMPLICATION FOR TROPICAL SPASTIC PARAPARESIS/HTLV‐I ASSOCIATED MYELOPATHY
Authors: MOR‐VAKNIN NIRIT , TURGEMAN HAVA , TORGEMAN AMRAM , WOLFSON MARINA , HULEIHEL MAHMOUD , ABOUD MORDECHAI
Published: September 2002

Tropical spastic paraparesis/HTLV‐I associated myelopathy (TSP/HAM), is characterized by infiltration of human T cell leukaemia virus type‐I (HTLV‐I)‐infected T‐cells, anti‐HTLV‐I cytotoxic T cells and macrophages into the patients’ cerebrospinal fluid and by intrathecally formed anti‐HTLV‐I antibodies. This implies that the disease involves a breakdown of the blood—brain barrier. Since astrocytes play a central role in establishing this barrier, the authors investigated the hypothesis that the HTLV‐I infected T cells disrupt this barrier by damaging the astrocytes. The present study revealed the HTLV‐I‐producing T cells conferred a severe cytopatic effect upon monolayers of astrocytoma cell line in co‐cultures. Following co‐cultivation, HTLV‐I DNA and proteins appeared in the monolayer cells, but after reaching a peak their level gradually declined. This appearance of the viral components was proved to result from a fusion of the astrocytic cells with the virus‐producing T cells, whereas their subsequent decline reflected the destruction of the resulting syncytia. This fusion could be specifically blocked by anti HTLV‐I Env antibodies, indicating that it was mediated by the viral Env proteins expressed on the surface of the virus‐producing cells. Similar fusion was observed between the HTLV‐I‐producing cells and certain other human nervous system cell lines. If such fusion of HTLV‐I‐infected T cells occurs also with astrocytes and other nervous system cells in TSP/HAM patients, it may account, at least partially, for the blood—brain barrier breakdown and some of the neural lesions in this syndrome.
LOCALIZATION OF ANNEXIN VI IN THE ADULT AND NEONATAL HEART
Authors: LUCKCUCK TRACY , TROTTER PATRICK J. , WALKER JOHN H.
Published: September 2002

Annexins are a major family of intracellular Ca2+‐binding proteins which have been implicated in a variety of cellular functions. In this paper the authors have used confocal microscopy to compare the distribution of annexin VI in vibratome sections of the rat adult left ventricle and striated muscle of the rat oesophagus. It is shown that in rat cardiac myocytes annexin VI is associated with only the sarcolemma and intercalated discs. In contrast, it is demonstrated that in rat skeletal muscle annexin VI is associated with the sarcoplasmic reticulum, in addition to the plasma membrane, suggesting that annexin VI is regulating different processes in these tissues. Also shown is that in vibratome sections of the neonatal rat left ventricle, annexin VI has a different subcellular location to that observed in the terminally differentiated adult myocyte. In these differentiating neonatal cells annexins VI is also associated with specific subcellular structures. Furthermore, using confocal microscopy of isolated myocytes the authors demonstrate that the association of annexin VI with the sarcolemma is stable even after cells are treated with the intracellular calcium chelator BAPTA‐AM, to greatly deplete cytosolic calcium levels. This demonstrates that annexin VI associates tightly with the sarcolemma, and suggests that components in addition to phospholipid are involved in binding annexin VI to the membrane. These results demonstrate that the subcellular location of annexin VI is differentially regulated, and suggest that annexin VI is required for a process or processes characteristic of the sarcolemma, and of the sarcoplasmic reticulum of skeletal but not of heart muscle.
ALTERATIONS IN DISTRIBUTION OF ACTIN BINDING PROTEINS IN UTERINE STROMAL CELLS DURING DECIDUALIZATION IN THE RAT
Authors: SHAW TIMOTHY J. , TERRY VERA , SHOREY CEDRIC D. , MURPHY CHRISTOPHER R.
Published: September 2002

Immunohistochemistry was used to investigate the involvement of the actin‐associated binding proteins, tropomyosin, α‐actinin and gelsolin with the formation of the decidual cell reaction during early pregnancy in the rat. Tropomyosin was present in the uterine myometrium, but absent from the both decidual and non‐decidual stromal cells. α‐Actinin was absent from non‐decidual stromal cells, but present in decidual cells. Gelsolin was present in non‐decidual cells close to the uterine stroma as well as in transformed decidual cells. Both gelsolin and α‐actinin were concentrated around the periphery of the cell. It is proposed that these actin‐binding proteins may be involved with the cellular transformations associated with decidualization.
AGE‐RELATED CHANGES IN THE DENDRITES OF OLFACTORY RECEPTOR NEURONS IN THE MALE SILKMOTH ANTHERAEA PERYNI (INSECTA, LEPIDOPTERA: SATURNIIDAE)
Authors: KUMAR G. L. , POPHOF B. , KUMAR S. , KEIL T. A.
Published: September 2002

A study was conducted to evaluate the changes that occur with aging at the dendrite level of the olfactory receptor neuron in the male silkmoth Antheraea pernyi. Using calcein AM/ethidium homodimer‐1 solutions, we found increased numbers of dendrites with damaged membrane with aging. Correspondingly there was an overall decrease in the electrophysiological activity as evidenced by the decreased number of cells discharging nerve impulse in response to female pheromone. It was also seen that the number of membrane swellings increased with age. In young animals aged 1–4 days, swellings showed intact membrane, and in older animals aged 5–15 days, they showed damaged membranes. With TUNEL assay that detects fragmented DNA in dying cells, an increased number of dendrites showing cytoplasmic labelling with age was found. The presence of fragmented DNA within aged dendrites was also confirmed in polyacrylamide gels after DNA extraction and PCR amplification. When tested for reversal of phosphatidylserine from the inner leaflet to the outer leaflet of plasma membrane no reactivity was seen. It appears that changes that occur during aging of dendrites may reflect some of the recognized symptoms of both apoptosis and necrosis.
PROLIFERATION AND COLLAGEN SYNTHESIS OF HUMAN ANTERIOR CRUCIATE LIGAMENT CELLS IN VITRO: EFFECTS OF ASCORBATE‐2‐PHOSPHATE, DEXAMETHASONE AND OXYGEN TENSION
Authors: Fermor B. , Urban J. , Murray D. , Pocock A. , Lim E. , Francis M. , Gage J.
Published: September 2002

Clinical and experimental studies demonstrate that injured anterior cruciate ligaments (ACL) do not usually heal and that autografts used to repair the ACL rapidly weaken in the early period and take a long time to regain strength. The aim of this study was to develop an in vitro culture system in which environmental and biochemical factors influencing the proliferation and matrix synthesis of cells derived from human anterior cruciate ligaments can be studied.

Primary cultures of human ACL cells were obtained by outgrowth from explants of normal ACL obtained at knee replacement for osteoarthritis in Dulbecco's minimum essential medium (DMEM). The effects of the additives 100μm l‐ascorbic acid‐2‐phosphate (Asc‐2‐P) and 10nmdexamethasone (dex) on proliferation and collagen synthesis were assessed after 4, 8 and 12 days in culture. Ligament cells were grown at 0, 5, 10 and 21% p O2in the presence of 100μmasc‐2‐P and 10nmdex. DNA content was assessed using the Hoechst dye method and collagen synthesis by the incorporation of 5mCi/ml [3H]proline after 3, 6 and 12 days in culture.

At 21% p O2, the presence of asc‐2‐P and dex induced significantly greater (P< 0.01, ANOVA) cell proliferation than with either additives alone. Greatest percentage collagen to total protein synthesis was observed when cells were grown in the presence of asc‐2‐P only. Least proliferation and percentage collagen to total protein synthesis was seen when both additives were omitted. Greatest cell proliferation was seen when cells were grown in 10% p O2and 5% p O2was associated with increased collagen synthesis.

These results suggest that it is possible to study the effects of environmental and biochemical factors on human ACL healing in vitro. Our data suggest oxygen can influence certain biosynthetic activities of ACL cells. Low oxygen tensions lead to an increase in collagen production by ACL cells. However early responses to injury require extensive cell proliferation which may be activated at higher p O2. Variation of p O2in ligaments during healing may therefore be an important modulator of successful repair.
STAUROSPORINE‐INDUCED CELL DEATH IN TETRAHYMENA THERMOPHILA HAS MIXED CHARACTERISTICS OF BOTH APOPTOTIC AND AUTOPHAGIC DEGENERATION
Authors: CHRISTENSEN SØREN T. , CHEMNITZ JOHN , STRAARUP ELLEN M. , KRISTIANSEN KARSTEN , WHEATLEY DENYS N. , RASMUSSEN LEIF
Published: September 2002

Staurosporine blocks signal transduction associated with cell survival, proliferation and chemosensory behaviour in the ciliated protozoan, Tetrahymena thermophila. Staurosporine inhibits cell proliferation and in vivo protein phosphorylation induced by phorbol ester. It also reduces the in vitro phosphorylation of the PKC‐specific substrate, myelin basic protein fragment 4‐14. Our results show that cell death in the presence of staurosporine is associated with morphological and ultrastructural changes similar to both apoptosis and autophagic degeneration, but these in turn can be postponed or prevented by 8‐bromo‐cyclic GMP, protoporphyrin IX, hemin or actinomycin D, although phorbol ester and insulin were ineffective. The results support the notion that staurosporine‐induced cell death is an active process, associated with and/or requiring de novo RNA synthesis.
X‐LINKED CHARCOT‐MARIE‐TOOTH DISEASE AND CONNEXIN32
Authors: Ionasescu Victor V.
Published: September 2002

We studied 29 families with X‐linked dominant CMT (CMTX1) neuropathy. Twenty‐five families showed mutations in the coding region of the connexin32 (Cx32) gene. The mutations included five nonsense mutations, 17 missense mutations, two medium size deletions and one insertion. Most missense mutations showed a mild clinical phenotype and slowing of motor nerve conduction velocities. All five nonsense mutations, the larger deletion and the insertion showed severe clinical phenotype. Four CMTX1 families with mild clinical phenotype showed no point mutations of the Cx32 gene coding region. Two mutations of the non‐coding region were identified. The first mutation was located in the nerve specific Cx32 promoter, the second mutation was located in the 5′ untranslated region of the mRNA.
GAP JUNCTIONAL COMMUNICATION IN THE DEVELOPING CENTRAL NERVOUS SYSTEM
Authors: Naus Christian C. G. , Bani‐Yaghoub Mahmud
Published: September 2002

The development of the central nervous system is a complex process involving multiple interactions between various cell types undergoing mitosis, migration, differentiation, axonal outgrowth, synaptogenesis and programmed cell death. For example, neocortical development is characterized by a series of transient events that ultimately leads to the formation of a discrete pattern of laminar and columnar organization. While neuron—glial cell—cell interactions have been shown to be involved in neuronal migration, recent observations that neurons are extensively coupled by gap junctions in the developing neocortex have implicated this phenomenon in the process of neocortical differentiation. The present review will examine the putative role of gap junctional intercellular communication in development of the central nervous system, with specific reference to recent studies in the development of the cerebral cortex.
REORGANIZATION OF MYOSIN AND FOCAL ADHESION PROTEINS IN SWISS 3T3 FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR BETA
Authors: Lee Kyung Mee , Park Jinseu , Kim Jae Hong , Yie Se Won , Chun Gie‐Taek , Kim Pyeung‐Hyeun , Choi Eui Yul
Published: September 2002

Certain types of cells show a dramatic change in cell morphology cultured in the presence of transforming growth factor beta (TGF‐β). To identify cellular components or factors leading to morphological changes, we investigated if any members of cytoskeletal proteins and cell‐adhesion molecules were redistributed in TGF‐β‐treated Swiss 3T3 fibroblasts by indirect immunofluorescence and Western‐blot analysis. Changes in cell morphology became apparent within 12h of the addition of TGF‐β and new RNA and protein synthesis was necessitated by the changes. While TGF‐β induced reorganization of microfilaments as reported in earlier studies, one of the actin isoforms, alpha actin of smooth muscle, was induced to form stress fibers in Swiss 3T3 cells. It was observed that myosin light chain was relocated from cell periphery to cytoplasmic filamentous structures by TGF‐β treatment, with an increased amount. In addition, the cell‐shape change was accompanied by an increase in the level of vinculin and tyrosine phosphorylation at focal adhesions. These results suggest that new protein synthesis is required for the cell‐shape change, and acto‐myosin filaments and focal adhesion proteins are involved in the alteration of cell morphology induced by TGF‐β in Swiss 3T3 fibroblasts.
MUTATIONS IN THE ATP‐BINDING DOMAIN AFFECT THE SUBCELLULAR DISTRIBUTION OF MITOTIC CENTROMERE‐ASSOCIATED KINESIN (MCAK)
Authors: Wordeman Linda , Wagenbach Mike , Maney Todd
Published: September 2002

Mitotic centromere‐associated kinesin (MCAK) is important for anaphase chromosome segregation. MCAK is diffusely localized to both the cytoplasm and the nucleus during interphase. At prophase MCAK is recruited to mitotic centromeres. It is associated with centromeres throughout mitosis and then returns to exhibiting a diffuse nuclear and cytoplasmic localization during interphase. MCAK has several predicted nuclear localization sequences. The subcelluar distribution of expressed deletion constructs of GFP‐MCAK suggest that the nucleocytoplasmic ratio of MCAK protein is dependent on a balance between several predicted nuclear localization sequences (NLS) and a putative nuclear exclusion sequence (NES) in the amino‐terminal region of MCAK. Amino acid substitutions in the ATP‐binding domain of the MCAK motor affect nuclear localization, which, in turn, influences the degree of centromere binding.
CHANGES IN PLASMA MEMBRANE FLUIDITY OF IMMORTAL RODENT CELLS INDUCED BY ANTICANCER DRUGS DOXORUBICIN, ACLARUBICIN AND MITOXANTRONE
Authors: Jedrzejczak Malgorzata , Koceva‐Chyla Aneta , Gwozdzinski Krzysztof , Józwiak Zofia
Published: September 2002

The aim of this study was to examine the effect of three structurally different anticancer drugs—the pro‐oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA‐DPH and 12‐AS) and spin labels (5‐DS and methyl‐12‐DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2–5μm). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20μm) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure.
TRANSIENT APOPTOSIS ELICITED BY INSULIN IN SERUM‐STARVED GLIOMA CELLS INVOLVES Fas/Fas‐L AND Bcl‐2
Authors: Yang Bei‐Chang , Wang Yuh‐Sheng , Wang Cheng‐Hsin , Lin Hsi‐Hui , Tang Ming‐Jer , Yang Tsae‐Lian
Published: September 2002

The expression of fas gene in glioma cells varies with growth stage. When insulin‐elicited transient apoptosis of glioma cells was in progress, the expression of fas gene increased at both transcriptional and translational levels. In contrast, the expression of fas‐L gene in glioma cells remained constant. Apoptosis occurred in the cells having high level of surface Fas protein. When the expression of Fas‐L in U‐373MG cells was suppressed by ribozyme, the insulin‐elicited transient apoptosis vanished. Overexpression of Bcl‐2 in U‐373MG cells did not alter significantly the cell cycle progression and the expression of fas gene. However, these cells were resistant to insulin‐trigged death. Therefore, insulin‐elicited apoptosis involved Fas‐related death signal, and which could be prevented by the protective effect of Bcl‐2.
GENDER‐RELATED DIFFERENCES IN ADHESION, GROWTH AND DIFFERENTIATION OF VASCULAR SMOOTH MUSCLE CELLS ARE ENHANCED IN SERUM‐DEPRIVED CULTURES
Authors: Bačáková L. , Mareš V. , Lisá V.
Published: September 2002

In 1‐day cultures with 10% serum, the number of rat aortic smooth muscle cells (VSMC) adhering to the growth support was similar in cells from both sexes, whereas in 1% serum, the number of VSMC from male donors was lower. In 10% serum medium, the doubling time was significantly shorter and the number of [3H]thymidine‐labelled nuclei was higher in cells of high passage from male rats. In serum‐free medium, these differences increased and were also seen in cells of low passage number. Morphologically, the cells in male‐derived cultures at higher passage number were mainly spindle‐shaped, formed well‐developed ‘hills and valleys’ and possessed longitudinally oriented bundles of α‐actin‐containing microfilaments. Most cells from female rats were flat, polygonal, the multilayered ‘hills’ were less prominent, with α‐actin microfilaments forming a mesh‐like network.
MOTOGENIC AND BIOSYNTHETIC RESPONSE OF ADULT SKIN FIBROBLASTS TO TGF‐β ISOFORMS (−1, −2 AND −3) DETERMINED BY ‘TISSUE RESPONSE UNIT’: ROLE OF CELL DENSITY AND SUBSTRATUM
Authors: Ellis I.R , Banyard J. , Schor S.L
Published: September 2002

We have recently demonstrated that the three principal mammalian isoforms of transforming growth factor β (TGF‐β) exert distinct effects upon: (1) the migration of confluent adult fibroblasts into 3D gels of native type I collagen fibres (i.e. TGF‐β−1 and −2 had no apparent motogenic activity, whilst TGF‐β−3 induced a dose‐dependent stimulation of cell migration); and (2) the synthesis of hyaluronan (HA) by these cells is also affected by the TGF‐β isoforms in a manner which parallels their effect on cell migration. The objective of the present study is to elucidate the manner in which this differential activity of the TGF‐β−1, −2 and −3 may be modulated by experimental parameters. Data presented in this communication indicate that cytokine bioactivity is determined by a combination of cell density and the nature of the macromolecular substratum. Thus, we now report that all three TGF‐β isoforms inhibit the migration of subconfluent cells in the collagen gel assay. Our data confirm that the migration of confluent cells is stimulated by TGF‐β−3 and further indicate that this motogenic activity is completely abrogated by either TGF‐β−1 or −2 when these are co‐incubated with TGF‐β−3. In contrast to these results obtained using a native type I collagen substratum, all three isoforms stimulated adult fibroblast migration in the transmembrane assay (in which cells are adherent to a 2‐D porous polycarbonate substratum). The precise effect of TGF‐β isoforms on HA synthesis was also affected by cell density and the nature of the substratum in a manner which paralleled their diverse effects on cell migration (i.e. stimulation, inhibition or no effect). Streptomyces hyaluronidase completely neutralized the TGF‐β−3‐induced stimulation of confluent fibroblast migration, thus suggesting a mechanistic link between the cytokine‐induced cell migration and HA synthesis under these conditions. Taken together, these data indicate that: (1) the bioactivity of TGF‐β−1, −2 and −3 are determined by cell density, the macromolecular substratum and the presence of other cytokines; and (2) it is therefore necessary to define cytokine bioactivity within the context of a larger ‘tissue response unit’ which more fully defines the activity state of the target cell and its microenvironment.
REACTIVE OXYGEN SPECIES INDUCE APOPTOSIS OF SYNOVIOCYTES IN VITRO. α‐TOCOPHEROL PROVIDES NO PROTECTION
Authors: Galleron S. , Borderie D. , Ponteziere Ch , Lemarechal H. , Jambou M. , Roch‐Arveiller M. , Ekindjian O.G , Cals M.J
Published: September 2002

Reactive oxygen species (ROS) are released during the inflammation of the synovial membrane associated with cartilage degradation in osteoarthritis. In this work, we exposed synoviocytes to superoxide anions at concentrations that may cause either apoptosis or necrosis. We studied membrane organization, dehydrogenase mitochondrial activity and nuclear morphology and integrity, to determine the nature of the death process initiated by superoxide anions and tried to counteract ROS effects with α‐tocopherol. We found that oxidative stress caused synoviocytes to undergo a process of cell death of an apoptotic nature rather than necrotic. Mitochondrial injury occurred at an early stage, and the FITC‐annexin‐V‐positive/propidium iodide‐positive cells occurred later than the metabolic changes. DNA strand breaks were evident at 8h and nuclear condensation at 24h. No LDH activity was detected in culture supernatants. In our experimental conditions, α‐tocopherol had little effect on stress damage; the antioxidant properties of this molecule did not affect the apoptosis caused by superoxide anions.
A GLYCOPROTEIN ISOLATED FROM THE SPONGE, PACHYMATISMA JOHNSTONII, HAS ANTI‐LEISHMANIAL ACTIVITY
Authors: Le Pape Patrice , Zidane Mohamed , Abdala Hiam , Moré Marie‐Thérèse
Published: September 2002

A high anti‐leishmanial activity was observed in an aqueous extract from the marine spongePachymatisma johnstonii, Bowerbank 1842 (Demospongiae, Geodiidae). Pachymatismin, a glycoprotein, was purified and shown to be a cytotoxic agent, which acts on promastigote and clinical‐like amastigote stages with IC50about 1μg protein/ml and induces changes in the cell shape, phospholipase A2activity and invasion capacity of the parasite. We believe pachymatismin is the first reported substance from a marine organism with anti‐leishmanial activity.
CD56 DIRECTLY INTERACTS IN THE PROCESS OF NCAM‐POSITIVE TARGET CELL‐KILLING BY NK CELLS
Authors: Takasaki Satoko , Hayashida Kazuhiro , Morita Chie , Ishibashi Hiromi , Niho Yoshiyuki
Published: September 2002

The role of CD56 in the process of target cell killing by NK cells has been investigated. Addition of NK cells to HuH28 cells, a CD56‐expressing cell line, led to inhibition of the growth of the target cells, which exhibited morphological features of apoptosis. These changes were prevented by the addition of a polyclonal anti‐NCAM to the cultures. Since neither Fas antigen expression nor apoptotic changes were induced by addition to a mixed culture supernatant of NK and target cells, both the Fas‐Fas ligand system and soluble factors do not seem to participate in apoptosis in these circumstances. Increased secretion of interferon‐γ and tumour necrosis factor‐α by NK cells must therefore have been suppressed by the presence of the polyclonal antibody. These results lead us to conclude that CD56, through homophilic binding, plays an important role in the process of target cell killing by an apoptosis mechanism.
CYTOKINESIS IN CELLS UNDERGOING MITOSIS WITHOUT GENOME REPLICATION
Authors: Wise Dwayne A.
Published: September 2002

Chinese hamster ovary cells can be forced to enter mitosis without prior DNA replication by treatment with hydroxyurea and caffeine. Cells treated in this way assemble a spindle that functions normally except that it does not accomplish anaphase spindle elongation (anaphase B). The chromatin detaches from the unreplicated kinetochores, which fragment, but establish microtubule attachments and migrate to the metaphase plate. Partitioning of the kinetochore fragments ensues on the normal schedule. Typical midbodies and cleavage furrows are established and daughter cells of equal size are produced. These results imply that intact chromosomes are not necessary for correct cleavage furrow placement but that kinetochores might be. Further, it is clear that cleavage furrow placement does not depend on anaphase spindle elongation.
UNILATERAL AND WANDERING FURROWS DURING MITOSIS IN VERTEBRATES: IMPLICATIONS FOR THE MECHANISM OF CYTOKINESIS
Authors: Savoian M.S , Khodjakov A. , Rieder C.L
Published: September 2002

Vertebrate somatic cells sometimes form unilateral furrows during cytokinesis that ingress from only one edge of the cell. In some cases after a cell initiates a normal symmetrical circumferential furrow, one of its edges stops furrowing and regresses while the furrow associated with the opposing edge continues across the cell. In cells containing two independent spindles unilateral furrows are sometimes formed that do not follow a linear path but instead sharply change their direction and wander for >40μm through the cell. These observations reveal that the ‘contractile ring’ normally seen during cytokinesis is composed of multiple independent ‘furrowing units’ that are normally coordinated to form a symmetrical furrow around the cell, and that once formed this so‐called contractile band does not function as a ‘purse string’ as commonly envisioned. Individual furrowing units can work independently of one another, and cytokinesis in vertebrates can be consummated by the formation of a single functional furrowing unit in a localized region of the cell cortex that is then propagated across the cell. How this propagation occurs remains an important question for the future.
HISTOCHEMICAL CHARACTERIZATION OF CELL DEATH IN HONEYBEE LARVAE MIDGUT AFTER TREATMENT WITH PAENIBACILLUS LARVAE, AMITRAZ AND OXYTETRACYCLINE
Authors: Gregorc Aleš , Bowen Ivor D.
Published: September 2002

A number of techniques were employed to assess cell death induced in honeybee larvae midgut afterper os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24h after treatment. Cell death reduced to 36% in the epithelial cells, 48h after treatment. In Oxytetracycline‐treated larvae, cell death was identified in 40% of midgut epithelial cells, 24h after inoculation and increased to 55% over the next 24h. In Paenibacillus ‐infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24h after Amitraz application. Cell death was reduced to 9% over the next 24h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.
PACKING OF MYOSIN MOLECULES IN MUSCLE THICK FILAMENTS
Authors: Miroshnichenko N. S. , Balanuk I. V. , Nozdrenko D. N.
Published: September 2002

The backbone of the myosin filament is an aggregate of α‐helical coiled coil myosin rods. Its surface forms a three‐stranded helix composed of myosin heads. Currently there is no adequate model to describe the organization of the myosin filament. It is proposed here that, in cross‐section the light meromyosin (LMM) of 18 myosin molecules form an outer tube, with nine S2 forming the interior core. At the surface of the thick filament, myosin heads are arranged in three rows, giving the filament a periodicity of 14.3nm per three myosin molecules. Two of these molecules are organized at an angle of 120 degrees to each other on the same level, while the third is shifted 7.2nm along the filament axis. This packing gives a striation pattern of 7.2nm by electron microscopy. An alternative model is also possible, in which the heads of the myosin molecules are uniformly spaced at an interval of 14.3nm along the filament axis. The packing of individual molecules within the myosin filament is based on a regular pattern of charge on the 28 amino‐acid repeat in the rod domain.
EFFECT OF ABSCISIC ACID AND COLD ACCLIMATION ON THE CYTOSKELETAL AND PHOSPHORYLATED PROTEINS IN DIFFERENT CULTIVARS OFTRITICUM AESTIVUML
Authors: Olinevich Olga V. , Khokhlova Ludmila P. , Raudaskoski Marjatta
Published: September 2002

In winter wheat, the tubulin and 60kDa‐phosphorylated proteins/actin ratio is considerably higher in the roots than in the leaves. Differences in the content of the main cytoskeletal proteins were also found in the leaves of the different cultivars. It is suggested that the lower amount of the tubulin and 60kDa‐phosphorylated proteins and higher content of actin determine the greater tubulin cytoskeletal stability in the leaves and their higher frost resistance, as compared with the roots. Also, it is possible that the higher content of the tubulin and 60kDa‐phosphorylated proteins defines the lower microtubule (MT) stability in the leaves of the low frost resistant cultivar than in the leaves of the more frost resistant ones. In the roots and leaves of the low frost resistant cultivar, the low stability of the numerous tubulin structures is apparently one reason for the abscisic acid (ABA)‐induced reduction of the cytoskeletal and 60kDa‐phosphorylated proteins in the cells. The cold acclimation compensated the ABA effect in the roots of the very frost resistant cultivar in the most extent. This suggests the existence of the different pathways in the increased plant cell frost resistance through the action of ABA and low temperature.
DIFFERENTIAL RESPONSIVENESS OF PROSTATIC ACID PHOSPHATASE AND PROSTATE‐SPECIFIC ANTIGEN mRNA TO ANDROGEN IN PROSTATE CANCER CELLS
Authors: Lin Ming‐Fong , Lee Ming‐Shyue , Garcia‐Arenas Renee , Lin Fen‐Fen
Published: September 2002

Androgens regulate the expression of both human prostatic acid phosphatase (PAcP) and prostate‐specific antigen (PSA), two major prostate epithelium‐specific differentiation antigens. Due to the important role of these two enzymes as prostate epithelium differentiation markers, we investigated their regulation of expression at the mRNA level in LNCaP human prostate carcinoma cells. Interestingly, phenol red, a pH indicator in the culture medium, promoted cell growth. To eliminate this non‐specific effect, a phenol red‐free, steroid‐reduced medium was utilized. When high‐density cells were grown in that medium, 5α‐dihydrotestosterone (DHT) suppressed PAcP but stimulated PSA. However, tumor promoter phorbol ester 12‐ o ‐tetradecanoyl phorbol‐13‐acetate (TPA) functioned as a potent inhibitor of both PAcP and PSA expression. Prolonged treatment with DHT as well as TPA resulted in a similar down‐regulation of protein kinase C and cellular PAcP activities. Thus, the levels of PAcP and PSA mRNA are differentially regulated by androgens in LNCaP cells.
HEAT SHOCK‐INDUCED ACTIN POLYMERIZATION, SAPK/JNK ACTIVATION, AND HEAT‐SHOCK PROTEIN EXPRESSION ARE MEDIATED BY GENISTEIN‐SENSITIVE TYROSINE KINASE(S) IN K562 CELLS
Authors: Han Song Iy , Ha Kwon‐Soo , Kang Kwang Il , Kim Han Do , Kang Ho Sung
Published: September 2002

Upon exposure to elevated growth temperatures, mammalian cells exhibit a variety of cellular responses, such as the expression of heat‐shock proteins (HSPs) and the activation of stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK). In this study, we show that heat shock transiently induces morphological change (cell elongation) and polymerization of actin, but not of microtubules, in human erythroleukaemic K562 cells. Pretreatment with actinomycin D or cycloheximide did not prevent the heat shock‐induced cell elongation and actin reorganization, indicating that gene transcription and protein synthesis are not required for this phenomenon. The alterations in cell morphology and actin structure in response to heat shock were specifically inhibited by genistein, a tyrosine kinase inhibitor, but not by other kinase inhibitors, including tyrosine kinase inhibitors (herbimycin and tyrphostin) and protein kinase C inhibitors (staurosporine and H7). The activities of genistein‐sensitive tyrosine kinase (GTK) and c‐Src were enhanced by heat‐shock treatment. In addition, a 75kDa protein was highly phosphorylated in its tyrosine residues(s) by heat shock, and the phosphorylation was prevented by genistein pretreatment. Genistein also inhibited the heat‐shock‐induced SAPK/JNK activation and HSP expression. In contrast, while colchicine, a microtubule‐disrupting agent, was able to induce actin polymerization and SAPK/JNK activation, these events were not inhibited by genistein. These results suggest that the heat‐shock‐induced actin polymerization, HSP expression, and SAPK/JNK activation may be mediated by the specific signal pathway involving GTK(s), while colchicine‐induced actin polymerization and SAPK/JNK activation is regulated in a different manner.
EFFECT OF BONE MORPHOGENETIC PROTEIN‐6 ON HAEMOPOIETIC STEM CELLS AND CYTOKINE PRODUCTION IN NORMAL HUMAN BONE MARROW STROMA
Authors: Ahmed N. , Sammons J. , Carson R. J. , Khokher M. A. , Hassan H. T.
Published: September 2002

Normal human bone marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and type II receptors. As a member of the TGF‐β super‐family, BMP‐6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP‐6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP‐6 on human cobblestone‐area‐forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long‐term bone marrow culture. Also, the effect of BMP‐6 on marrow stroma production of interleukin‐6, −11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri‐lineage differentiation was examined. Moreover, the effect of BMP‐6 on marrow stroma release of soluble adhesion molecule VCAM‐1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP‐6 treatment from 88±10 per 105cells in control cultures in a dose dependent manner to only 48±3 per 105cells in 50ng/ml BMP‐6‐treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50ng/ml BMP‐6 was able to significantly reduce IL‐6 and IL‐11 production from marrow stroma, P< 0.01. Also, BMP‐6 significantly increased soluble gp130 release by 7.4‐fold in 50ng/ml BMP‐6‐treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL‐6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM‐1 released increased by two‐fold in 50ng/ml BMP‐6‐treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM‐1 (ligand for VLA4). Recently, blocking the VLA4/VCAM‐1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G‐CSF‐mobilised blood CD34 positive cells than on bone marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for bone repair and fracture healing, the present results suggest a possible role for BMP‐6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP‐6 in human long‐term bone marrow culture.
ENVIRONMENTAL MODULATION OF αv, α2AND β1INTEGRIN SUBUNIT EXPRESSION IN HUMAN OESOPHAGEAL SQUAMOUS CELL CARCINOMAS
Authors: Miller Sharon E. , Veale Rob B.
Published: September 2002

Integrins are cell adhesion molecules pivotal in regulating normal cell behaviour. Ectopic expression of integrins, characteristic of transformed cells, is instrumental in differentiation, proliferation, apoptosis, angiogenesis, matrix degradation and migration. Oesophageal squamous cell carcinoma (SCC) has a propensity to metastasize and hence an extremely poor prognosis. It is shown here that oesophageal SCCs express αvstrongly and that normal oesophageal tissue does not express αv. This makes αva significant indicator of the transformed phenotype. α2and β1integrin subunits are down‐regulated in oesophageal SCCs compared to normal oesophagus. Dominance of the α2β1heterodimer is symptomatic of potential loss of other β1binding integrins in oesophageal SCCs. These results suggest a decrease in rigid cell adhesion possibly increasing migratory potential, whilst simultaneously permitting the adhesion and migration of SCC cells on a large repertoire of ligands due to de novo αvexpression.
BIFUNCTIONAL EFFECTS OF HEPARIN‐BINDING PROTEIN HBp17 ON DNA SYNTHESIS IN CELLS
Authors: Chen Jun‐Hui , Wang Xin Chang , Sato J. Denry
Published: September 2002

A 17kD heparin‐binding protein (HBp17) has a biphasic dose‐dependent effect on DNA synthesis in 3T3 cells. Maximal stimulation of DNA synthesis occurs at 8ng/ml HBp17, but a half‐maximal inhibition occurs at ∼500ng/ml. This inhibition can easily be reversed by addition of 400pg/ml aFGF or 100pg/ml bFGF, whereas EGF had no effect. This biphasic action of HBp17 was also seen in human umbilical vein endothelial cells (HUVEC), whereas it was not found in the malignant cell line, A431‐AJC. The functional relationship between HBp17 and FGF is discussed.
GROWTH INHIBITION AND DIFFERENTIATION OF C6 GLIOMA CELLS ON TREATMENT WITH HMBA
Authors: Shirsat Neelam V. , Kayal Jyoti J. , Shaikh Sadaf , Mehta Ashish
Published: September 2002

HMBA, a differentiation inducer belonging to the class of hybrid polar compounds, is known to induce terminal differentiation of a number of leukemic and solid tumour cell lines. In this report we have shown that HMBA markedly inhibits growth of C6 glioma cells at non‐cytotoxic concentrations ranging from 2.5m m to 10m m in a dose‐dependent manner. The growth inhibitory effect can be detected as early as 18–24h. By the sixth day the growth inhibition decreases at all the concentrations tested. Treatment with HMBA results in an accumulation of C6 cells in G0/G1 phase along with a decrease in the number of cells in S phase. HMBA induces morphological differentiation of C6 cells and increases expression of glial fibriliary acidic protein (GFAP), a marker for mature astrocytes. HMBA induces c‐fos and represses cycloheximide‐induced c‐jun and fra‐1 expression. HMBA‐induced growth inhibition of C6 cells is accompanied by a decrease in Cdk4 protein levels. However, HMBA fails to sustain low Cdk4 levels, which may be responsible for HMBA's failure to sustain the growth inhibitory effect.
REGULATION OF GELATINASE‐A (MMP‐2) PRODUCTION BY OVINE INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS GROWN IN ALGINATE BEAD CULTURE BY TRANSFORMING GROWTH FACTOR‐β1AND INSULIN LIKE GROWTH FACTOR‐I
Authors: Pattison Scott T. , Melrose James , Ghosh Peter , Taylor Thomas K. F.
Published: September 2002

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell‐cell and cell‐matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP‐2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP‐2 by Transforming Growth Factor‐β1 (TGF‐β1) and Insulin‐like Growth Factor‐1 (βIGF‐I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum‐free conditions for 24h, then stimulated with TGF‐β1 (0.1 or 10ng/ml) or IGF‐I (2 or 50ng/ml) ±Concanavalin A (20μg/ml) for an additional 48h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP‐2) and Membrane Type 1 Matrix Metalloproteinase (MT1‐MMP) were immunolocalised in beads. Pro (72kDa) and active (59kDa) MMP‐2 were the major gelatinolytic MMPs detected in control cultures, the TGF‐β1 and IGF‐I treatments significantly decreased levels of the active MMP‐2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF‐I, and to a lesser extent with TGF‐β1. Cell surface levels of TIMP‐2 and MT1‐MMP were decreased by the TGF‐β1 treatment while IGF‐I only appeared to decrease TIMP‐2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.
CHARACTERIZATION AND REGULATION OF CONSTITUTIVE TRANSPORT INTERMEDIATES INVOLVED IN TRAFFICKING FROM THE TRANS‐GOLGI NETWORK
Authors: McLauchlan Hilary J. , James John , Lucocq John M. , Ponnambalam Sreenivasan
Published: September 2002

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans‐Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans ‐Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. Anin vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200nm in length. These membrane‐enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.
EXPRESSION OF BAX IN CELL NUCLEUS AFTER EXPERIMENTALLY INDUCED APOPTOSIS REVEALED BY IMMUNOGOLD AND EMBEDMENT‐FREE ELECTRON MICROSCOPY
Authors: Gajkowska Barbara , Motyl Tomasz , Olszewska‐Badarczuk Hanna , Godlewski Michał Marek
Published: September 2002

The unique combination of immunocytochemistry with embedment‐free electron microscopy was applied for precise and specific localisation of BAX in the human colon adenocarcinoma COLO 205 cell line stimulated to undergo apoptosis by camptothecin (DNA topoisomerase I inhibitor). Camptothecin‐induced apoptosis was associated with redistribution of BAX from cytosol to organelle membranes: mitochondria, Golgi apparatus, endoplasmic reticulum and via nuclear envelope pores to the nucleus, occurring within 60–180min of cell exposure to the drug. An increase in BAX immunoreactivity on fine filaments and the lamina‐pore complex of the nuclear matrix was also observed. The increase in BAX expression in the nuclear area of camptothecin‐treated COLO 205 cells was confirmed by quantitative analysis using laser scanning cytometry. The subcellular translocations of BAX preceded the appearance of any morphological symptoms of apoptosis.
ESTROGEN DOWNREGULATES THE NUMBER OF CAVEOLAE AND THE LEVEL OF CAVEOLIN IN UTERINE SMOOTH MUSCLE
Authors: Turi Agnes , Kiss Anna L. , Müllner Nandor
Published: September 2002

Estrogen and progesterone, while regulating uterine functions, also regulate the number of caveolae and the level of caveolin. Large numbers of caveolae, as well as elevated expression of caveolin‐1 and caveolin‐2 isoforms in the myometrium of ovariectomised (OVX) rats were detected. 17β‐estradiol (E2) has a downregulating effect: the treatment of OVX rats with E2 (5μg/animal) reduced the formation of caveolae by approx. 90%. Western blots clearly demonstrated the reduction of membrane caveolin‐1 and −2 content. Progesterone treatment (2.5mg/animal) alone did not cause any substantial change, but prevented the effect of estrogen. Control experiments showed that the quantity of Na+/K+‐ATPase, a plasma membrane protein excluded from caveolae, was not downregulated by E2. The administration of the pure estrogen receptor (ERα) antagonist ICI 182,780 (1mg/animal) not only compensated for the inhibitory effect of E2, but further increased the level of caveolin‐1 in the myometrium of OVX rats and facilitated the formation of caveolae by ∼70%. In contrast, the partial antagonist tamoxifen (1mg/animal) mimicked the effect of estrogen. The amount of caveolin also changed during pregnancy. During the first half of pregnancy the expression of caveolin was suppressed, but it gradually increased until delivery. Our results indicate that the formation and number of caveolae are influenced by the physiological state of the uterus in a hormone dependent manner.