bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophil polarisation in plasma differs to that induced by endogenous chemoattractants with regard to frequency of uropod formation and requirement for divalent cations.
Authors: Harkin Damien G. , Bignold Leon P.
Published: September 2002

Human neutrophils suspended in Hanks' balanced salt solution (37° C, 20 mM Hepes, pH 7.2) produced extensions, elongated and developed a polarised morphology with both a pseudopod and uropod when exposed to C5a (10 nM), leukotriene B4 (10 nM), platelet activating factor (40 nM) or interleukin‐8 (12.5 nM). Responses to each mediator were generally enhanced or unaffected by chelators of extracellular Ca2+ and Mg2+. Neutrophils suspended in heparinised plasma (90‐10% v/v in Hanks' balanced salt solution) produced extensions, elongated and developed a pseudopod, but rarely developed a uropod unless additional Mg2+ ions (0.5‐5 mM) were added. These findings demonstrate that the polarisation of neutrophils in plasma is significantly different to that induced by endogenous chemoattractants with regard to the frequency of uropod formation and requirement for extracellular divalent cations.
Reproduction of nuclei in Pelomyxa palustris
Authors: Daniels Edward W. , Pappas George D.
Published: September 2002

Light and electron micrographs were made of nuclei in Pelomyxa palustris, a unicellular, multinucleated giant amoeboid organism. We analyzed 1019 pelomyxae and classified their nuclei according to their location in the nuclear cycle. The majority of organisms (56.3%) had interphase nuclei, some of which contained spores of mostly 1‐3 μm in diameter. The nuclei had disintegrated in 1.3 % of organisms that appeared to have no nuclei. The remainder (42.4%) had nuclei in the form of spores (1 to 10 μm spheroids) that were in various stages of development and growth. Mitotic figures were seen in some of them, with several chromosome pairs per nucleus. Interchromosomal fibers were seen at anaphase, and newly formed "young" interphase nuclei were observed.
Retinoic acid induces changes in Xenopus embryo glycolipid pattern
Authors: Rizzo Angela M. , Gornati Rosalba , Rossi Federica , Bernardini Giovanni , Berra Bruno
Published: September 2002

Retinoic acid (RA), known for its important role in cellular differentiation, may cause a modification of glycolipid distribution characterized by a shift from globoserie towards latto‐ and ganglio‐series. In the present paper, we have investigated the modifications of the lipidic pattern after exogenous RA treatment of Xenopus embryos. We have noticed a decrease in neutral glycolipids with a parallel increase in gangliosides; the content of sulfatides does not seem to be modified. Beside the shift toward ganglio‐serie, we have also observed a redistribution inside this class of lipids. In particular, following RA treatment, the relative distribution of GD1b and GT1b increases while that of GM3 decreases.
Adipocyte morphology during hormone‐induced lipid deposition and mobilization: An ultrastructural investigation in the perfused cardiac fat
Authors: Zancanaro C. , Poltronieri R. , Sbarbati A. , Merigo F. , Cevese A.
Published: September 2002

The rat pericoronary adipose tissue was perfused in the presence of either the liposynthetic hormone insulin or the lipolytic hormone noradrenaline. Insulin perfusion associated with a) larger adipocyte mean sectional diameter in comparison with noradrenaline perfusion; b) glycogen deposition; c) appearance of small fat globules at discrete sites at the periphery of the main lipid drop. The two latter phenomena were apparently dose‐dependent. Massive lipid deposition was induced by addition of triglycerides to the perfusion medium and this associated with appearance of prominent endoplasmic reticulum in the cytoplasm. In noradrenaline‐perfused adipose tissue many small lipid droplets surrounded the central lipid deposit and the endoplasmic reticulum was in the form of both thin long, dashed cisternae sometime surrounding lipid droplets and grouped, anastomosing tubular cisternae. The present work shows that the perfused white adipose tissue of the heart is a suitable model to study, in situ, the morphological effects of hormones in adipocytes.
Achievement of thermotolerance trough hsps phosphorylation in sea urchin embryos.
Authors: Roccheri M. C. , Isola M. G. , Bosco L. , Cascino D. , Giudice G.
Published: September 2002

TPA treatment of sea urchin embryos is able to induce thermotolerance. Evidence is provided that TPA treatment induces phosphorylation of a constitutive stress protein of 38 KDa.
THERAPEUTIC STRATEGIES FOR THE INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE—POTENTIAL FOR A NOVEL CLASS OF ANTI‐INFLAMMATORY AGENTS
Authors: PFEILSCHIFTER JOSEF , EBERHARDT WOLFGANG , HUMMEL RICHARD , KUNZ DIETER , MÜHL HEIKO , NITSCH DOROTHEA , PLÜSS CHRISTOPH , WALKER GABY
Published: September 2002

In recent years, NO, a gas previously considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell—cell communication throughout the body. In mammals, NO is a recognized mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a non‐conventional neurotransmitter and participates in the establishment of long‐term plasticity required for memory formation. In addition, NO is responsible for some parts of the host response to sepsis and inflammation and contributes to certain disease states. A number of strategies have emerged with regard to a pharmacological control of pathological NO overproductions. This review will discuss these novel therapeutic approaches that may provide new means for clinical medicine.
BREFELDIN A DISTORTS DISCOID AND POLARIZED MORPHOLOGICAL ORGANIZATIONS OF EPITHELIAL CELLS IN CULTURE
Authors: PRIGOZINA N. L. , DUGINA V. B. , VASILIEV J. M.
Published: September 2002

Brefeldin A (BFA), an agent disorganizing organelle traffic, was used to find out the role of this traffic in the maintenance of shape and cytoskeletal organization of cultured epithelial MDCK cells. Cell shape was assessed using dispersion and elongation indices (Dunn and Brown, 1986,J Cell Sci83: 313–340). BFA reversibly transformed discoid MDCK cells with circular actin pattern into moderately polarized cells with straight actin bundles. In other experiments the same MDCK cells were first transformed into highly polarized fibroblast‐like cells by incubation with ‘scatter factor’ (HGF/SF) and then treated with BFA. In these conditions BFA significantly decreased the degree of polarization. It is suggested that both highly polarized and discoid morphological organizations are controlled by organelle traffic.
DEMONSTRATION OF ALKALINE PHOSPHATASE PARTICIPATION IN THE MINERALIZATION OF OSTEOBLASTS BY ANTISENSE RNA APPROACH
Authors: TORII YASUYOSHI , HITOMI KIYOTAKA , YAMAGISHI YASUKO , TSUKAGOSHI NORIHIRO
Published: September 2002

MC3T3‐E1 cells grown with ascorbic acid express sequentially osteoblastic marker proteins such as alkaline phosphatase (ALPase) and then form a mineralized extracellular matrix (ECM) as a consequence of osteoblastic differentiation. To explore the functional roles of ALPase in the process of osteoblastic maturation, an inducible expression vector for antisense ALPase RNA was constructed and stably transfected into MC3T3‐E1 cells. The expression of antisense ALPase RNA in the differentiated MC3T3‐E1 transfectants reduced markedly the ALPase activity, which resulted in a significant decrease in the deposition of minerals upon prolonged culture. These findings demonstrated directly that ALPase participated in the mineralizationof ECM.
INTEGRIN SYNTHESIS AND UTILIZATION IN CULTURED HUMAN OSTEOBLASTS
Authors: PISTONE M. , SANGUINETI C. , FEDERICI A. , SANGUINETI F. , DEFILIPPI P. , SANTOLINI F. , QUERZÉ G. , MARCHISIO P. C. , MANDUCA P.
Published: September 2002

This study describes the adhesion of human osteoblasts, culturedin vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77–100%, in 2h and at 55nmsubstrata concentration, and it was accompained by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the β1 integrin and antibodies to the α5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against α2, α3, α5, αv, β1 andβ3 integrins we detected synthesis of α3β1, α5β1, αvβ3, and an αvβ1‐like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus α4, α1 and β5 subunits. In cells adhering in the presence of serum we showed organization of β3 and αv integrins in focal contacts. In cells adhering to fibronectin α5 and β1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.
HIGH FREQUENCY OSCILLATIONS IN THE ACTIVITY OF PHOSPHOTYROSINE PHOSPHATASE IN MURINE ERYTHROLEUKAEMIC CELLS: ACTION OF INSULIN AND HEXAMETHYLENE BISACETAMIDE
Authors: FERREIRA G. M. N. , WÖLFLE H. , HAMMOND K. D. , GILBERT D. A.
Published: September 2002

We have previously reported oscillations in the activities of the phosphoamino acid phosphatases in murine erythroleukaemic cells. In keeping with our predictions we now show that the phosphotyrosine phosphatase activity rhythm has a much shorter period than originally seemed the case, being of the order of 10min and probably less. The periodic changes show evidence of rhythmic modulation of mean, period and amplitude as with all other cellular oscillations studied. Insulin decreases the frequency of the rhythm while the inducer of differentiation, hexamethylene bisacetamide (HMBA) decreases its amplitude. Current ideas on phosphorylation dynamics in relation to metabolism and mitosis may need to be revised in the light of the observations.
PROGRAMMED CELL DEATH IN THE HONEY‐BEE (APIS MELLIFERA L.) LARVAE MIDGUT
Authors: GREGORC ALEŠ , BOWEN IVOR D.
Published: September 2002

The histochemical and cytochemical localization of acid phosphatase has been used in an attempt to map the sites of cellular lysis and death. Reaction product was found both in the brush border of the midgut epithelium and in the basal membrane. Vacuolar acid phosphatase activity was found in the regenerative epithelial cells. Extra‐cisternal reaction product was associated with the endoplasmic reticulum which was dilated in lysed areas of the cytoplasm. Free acid and alkaline phosphatase activity was found in the basal area of the midgut epithelial cells and the former also occurred in the haemocoel. In the tracheoblastic cells only vacuolar acid phosphatase activity was seen. Chromatin aggregates were distributed throughout the nucleus and the nuclear envelope showed some infolding. Certain mature epithelial cells proved positive for anti‐histone associated DNA fragmentation indicative of programmed cell death.
EXTRA‐LYSOSOMAL PROTEOLYSIS AND EXPRESSION OF CALPAINS AND CALPASTATIN IN CULTURED THYROID CELLS
Authors: KARLSSON J.‐O. , NILSSON M.
Published: September 2002

Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl‐Leu‐Tyr‐7‐amino‐4‐methylcoumarin. The Ca2+‐independent proteolytic activity was largely inhibited by substances known to affect cysteine‐ and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine‐ and aspartic proteases. Addition of Ca2+did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chomatography and Western blotting demonstrated the presence of both m‐calpain (40% of total calpain) and μ‐calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m‐calpain had a diffuse cytoplasmic distribution whereas μ‐calpain was located both in the cytoplasm and at the cell—cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.
INHIBITION OF MALIGNANT CELL PROLIFERATION BY CULTURE MEDIA CONDITIONED BY CARDIAC OR SKELETAL MUSCLE
Authors: ZINMAN TOVA , SALZBERG SAMUEL , MALIK ZVI , SHAINBERG ASHER
Published: September 2002

The present work is an attempt to explain the high resistance of muscles to cancer development. We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells. The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells. Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM. For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium). The concentrated sample was applied to a Sephadex G‐50 column and three fractions were obtained. Only one fraction showed inhibitory activity. Four protein bands were observed in this fraction, as revealed by SDS‐PAGE. We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.
EXPRESSION AND CELLULAR LOCALIZATION OF THE mRNA FOR THE 25‐kDa HEAT‐SHOCK PROTEIN IN THE MOUSE
Authors: WAKAYAMA TOMOHIKO , ISEKI SHOICHI
Published: September 2002

The 25‐kDa heat‐shock protein (Hsp25) is a member of the small heat‐shock protein family but its function remains largely unknown. In the present study we examined the expression and cellular localization of Hsp25 mRNA in mice under physiological, unstressed conditions using Northern blot and in situ hybridization analyses with specific oligonucleotide probes. At the organ level, high amounts of Hsp25 mRNA were detected in the oesophagus, skin, eye, stomach, lung and urinary bladder, with moderate amounts in the heart, skeletal muscle, aorta, adrenal gland, ovary, testis, uterus, large intestine, and thymus. At the cellular level, intense to moderate signals for Hsp25 mRNA were localized in the muscle cells of smooth, heart and skeletal types, in the epithelial cells of stratified squamous and transitional types and of the oviduct, in the steroid endocrine cells of the adrenal cortex and corpus luteum, as well as in the spermatocytes of the testis. In contrast, the signal was scarcely detectable in the nervous tissues, lymphatic tissues, the columnar epithelial cells of the digestive tract, or the parenchymal cells of the liver, pancreas and kidney. These results suggest some significant role for Hsp25 in distinct populations of mouse cells under physiological conditions.
APOPTOSIS INDUCED BY FILARIAL PARASITIC SHEATH PROTEIN IN HEp 2 CELL LINES BLOCKED BY ECTOPIC EXPRESSION OF bcl 2
Authors: KRISHNAMOORTHY BHAVANI , RENNER WOLFGANG , BALAKRISHNAN ARUN
Published: September 2002

Chronic filarial patients exhibit an occult manifestation, Tropical Pulmonary Eosinophilia, (TPE), caused by an exaggerated immune response to shed and circulating filarial antigens, leading to extensive lung damage. We have attempted to examine the disease in vitro using the human epithelial cell line, HEp2. Filarial sheath proteins induce apoptosis in HEp2 cells characterized by chromatin condensation, internucleosomal DNA cleavage, positive staining for TUNEL assay and shows a sub‐G1 peak on FACS analysis. In order to understand subcellular events and to analyse the protective role of bcl2, we engineered HEp2 to overexpress Bcl2 protein. HEp2 bcl2 cells do not undergo apoptosis on exposure to filarial sheath protein, indicating that filarial protein‐induced apoptosis in epithelial cells proceeds via a pathway, inhibitable by overexpression of bcl 2.
PARTICIPATION OF TWO ISOFORMS OF C/EBPβ TRANSCRIPTION FACTOR IN THE ACUTE‐PHASE REGULATION OF THE RAT HAPTOGLOBIN GENE
Authors: Grigorov Ilijana , MilosavljeviIć Tanja , Cvetković Ivana , Petrović Miodrag
Published: September 2002

Previous analyses of the mechanism of the transcriptional induction of the rat haptoglobin (Hp) gene during acute‐phase (AP)‐reaction have revealed the involvement of several trans ‐acting nucleoproteins (NPs) in controlling this process. In this study, by using antibodies against C/EBPβ factor in Western immunoblot assay, we found that rat liver trans ‐acting NPs p35 and p20 are two characteristic C/EBPβ isoforms whose expression is induced under AP‐conditions. DNA‐binding assays identified the binding sites for these two C/EBPβ proteins in the functionally defined elements A and C of the rat Hp gene and also revealed that they have specific binding affinity towards these elements. Under non‐induced conditions, p35 was the only C/EBPβ binding factor; however, upon AP‐conditions both, 35kDa‐ and 20kDa‐C/EBPβ binding activities were significantly induced suggesting that these interactions are necessary for the activation of the Hp gene. By in vitro phosphorylation assay and selective proteolysis, we also present evidence that p35 requires phosphorylation for its DNA binding ability. Thus, we conclude that increase in binding of C/EBPβ isoforms during AP‐reaction occurs through their upregulation and structural modification.
A STUDY OF THE EFFECT OF THEOPHYLLINE ON THE ANTI‐INFLAMMATORY POTENCY OF DEXAMETHASONE USING α‐AMINOISOBUTYRIC ACID UPTAKE IN RAT SKIN FIBROBLASTS: A POSSIBLE MECHANISM THROUGH THE GLUCOCORTICOID RECEPTOR
Authors: Xenos K. , Kalogeromitros D. , Markantonis Sophia L. , Katsambas A. , Katsarou Alexandra
Published: September 2002

The action of theophylline on the uptake of α‐aminoisobutyric acid (AIB; an indicator of anti‐inflammatory potency) stimulated by the glucocorticoid, dexamethasone, in cultured rat fibroblast monolayers was evaluated. Theophylline alone (0.1mm) did not show significant activity (3314±27cpm) compared with the baseline level (3186±130cpm), but in the presence of 10nmdexamethasone the stimulation of AIB uptake was increased to 5263±100cpm, approximately to the same extent as with 100nmdexamethasone alone (5397±28cpm). Activation of glucocorticoid‐receptor complexes in rat fibroblast cytosol was studied by assessing the extent of their binding to DNA‐cellulose. Activated and non‐activated forms of glucocorticoid‐receptor complexes were analysed by DEAE‐Sephadex chromatography. Theophylline (1mm) was found to have a direct effect (0°C), similar to that of heat (25°C) on DNA‐cellulose binding, that is approximately 64.5% and 68.7%, respectively, thus indicating that theophylline promotes activation of glucocorticoid receptors at low temperature. The effect of theophylline on the stimulation of AIB uptake by dexamethasone when considered in the light of its activation of GR receptors in the fibroblast cytosol indicates that this effect may be mediated by GR activation.
SHORT COMMUNICATIONSTEROID HORMONE (HYDROCORTISONE, OESTRADIOL AND TESTOSTERONE) UPTAKE, STORAGE OR INDUCED SYNTHESIS INTETRAHYMENA
Authors: Csaba G. , Poteczin Éva , Fehér T. , Kovács P.
Published: September 2002

After cyclodextrin‐coated 10−6m steroid hormone treatment for 3 days (hormonal imprinting), Tetrahymena cells and their media were analysed by radioimmunoassay for the same hormone and for the presence of the other two. In the absence of hormone treatment, the cells contained no detectable levels of the three steroids. By 2 days in fresh medium following exposure of cells to a 72h pretreatment of each specific hormone, correspondingly high quantities of hydrocortisone and oestradiol, but lesser quantities of testosterone, were found in both the media and the cells. One week after treatment only traces of hydrocortisone were found, exclusively within the cells themselves. Oestradiol was present in measurable quantities in both cells and media, whereas testosterone was only present in the medium. The presence of the other two hormones to the one used in the pretreatment were not usually present, except that when testosterone had been given, some oestradiol was also detected at 48h, suggesting Tetrahymena has a functional cytochrome P450aromatase.
THE TETRAHYMENA HOMOLOG OF BACTERIAL AND MAMMALIAN 4‐HYDROXYPHENYLPYRUVATE DIOXYGENASES LOCALIZES TO MEMBRANES OF THE ENDOPLASMIC RETICULUM
Authors: Neve Søren , Tornehave Ditte , Corydon Thomas Juhl , Kristiansen Karsten
Published: September 2002

The expression and intracellular localization of the Tetrahymena homolog of 4‐hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild‐type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS‐1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild‐type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis ofTetrahymena HPPD takes place in cells starved for more than 30min, these results suggest that there is a flow ofTetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and thatTetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.
COMBINED IMMUNOFLUORESCENCE AND FIELD EMISSION SCANNING ELECTRON MICROSCOPE STUDY OF PLASMA MEMBRANE‐ASSOCIATED ORGANELLES IN HIGHLY VACUOLATED SUSPENSOR CELLS OF WHITE SPRUCE SOMATIC EMBRYOS
Authors: Fowke Larry , Dibbayawan Teresa , Schwartz Owen , Harper John , Overall Robyn
Published: September 2002

Highly vacuolated suspensor cells of spruce somatic embryos were examined by immunofluorescence light microscopy using butyl‐methyl‐methacrylate (BMM) and polyethylene glycol (PEG) embedded sections, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). The use of PEG embedded embryos provided a rapid method for light microscope detection of antigens before committing to FESEM analysis. BMM embedded specimens provided well preserved suspensor cells for immunofluorescence. FESEM permitted high resolution observation of large areas of the inner surface of the plasma membrane and associated cell organelles. Suspensor cells contained mostly transversely oriented cortical microtubules linked to the plasma membrane and adjacent microtubules by cross‐ bridges. Light and electron microscopy revealed numerous clathrin coated structures on the plasma membrane. These included flat patches of clathrin, coated pits and coated vesicles. Many coated vesicles were associated with microtubules. Both tubular and lamellar endoplasmic reticulum were observed on the plasma membrane by FESEM.
MODULATION OF A2AADENOSINE RECEPTOR(S) BY K+ATPCHANNELS IN BOVINE BRAIN STRIATAL MEMBRANES
Authors: Olanrewaju Hammed A. , Marala Ravi B. , Mustafa S. Jamal
Published: September 2002

The modulation of adenosine receptor with K+ATPchannel blocker, glibenclamide, was investigated using the radiolabeled A2A‐receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration‐dependent manner with a maximum displacement being approximately 65% at 10−4m. In the presence of 10−5m glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10−4m. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd=10.6±1.71n m; Bmax=221.4±6.43fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10−5m glibenclamide; (Kd=1.3± 0.22n m; Bmax=74.3±2.14fmol/mg protein; and Kd=8.9±0.64n m; Bmax=243.2±5.71fmol/mg protein), indicating modulation of adenosine A2Areceptors by glibenclamide. These studies suggest that the K+ATPchannel blocker, glibenclamide, modulated the adenosine A2Areceptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that in the presence of glibenclamide it binds to two sites. These studies provide further insight into the interactions between K+ATPchannels and adenosine receptors.
BIOCHEMICAL AND IMMUNOCYTOCHEMICAL ANALYSIS OF VITELLOGENESIS IN THE OLIVE FRUIT FLY DACUS (BACTROCERA) OLEAE (DIPTERA: TEPHRITIDAE)
Authors: Trougakos Ioannis P. , Lamnissou Klea , Margaritis Lukas H.
Published: September 2002

Synthesis and selective accumulation of the major yolk proteins in the developing oocytes of the speciesDacus oleae (Diptera: Tephritidae) was studied biochemically and by immunoelectron microscopy. In the hemolymph of adult females, two yolk proteins precursors (or vitellogenins) have been detected. They each exhibit a similar molecular weight and isoelectric point to their respective mature yolk proteins (or vitellins), while electrophoretic analysis of their synthetic profile shows that their levels in the hemolymph increase rapidly during development. Immunogold electron microscopy of ovarian sections, revealed that the hemolymph vitellogenins reach the oocyte through enlarged inter‐follicular spaces and demonstrated vitellogenin synthesis by the follicle cells of the vitellogenic follicles. The newly synthesized vitellogenins follow a distinct secretory pathway into these cells as compared to other components being synthesized at the same time (e.g. the vitelline envelope proteins), since they were found in secretory vesicles that appeared to be differentiated from those destined to participate in the vitelline envelope. The vitellogenin‐containing vesicles exocytose their contents directionally into the follicle cell/vitelline envelope boundary, and subsequently the vitellogenins diffuse among the gaps of the forming vitelline envelope and reach the oocyte plasma membrane. Their internalization by the oocyte includes the formation of an endocytic complex consisting of coated pits, coated vesicles, endosomes, transitional yolk bodies, and finally mature yolk bodies, in which the storage of the vitellins and other yolk proteins occur. These results are discussed in relation to data obtained from other Dipteran species.
ANNEXIN V RELOCATES TO THE PERIPHERY OF ACTIVATED PLATELETS FOLLOWING THROMBIN ACTIVATION: AN ULTRASTRUCTURAL IMMUNOHISTOCHEMICAL APPROACH
Authors: Tzima Eleni , POUJOL Christel , Nurden Paquita , Nurden Alan T. , Orchard Margaret A. , Walker John H.
Published: September 2002

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1%±0.78 of annexin V is present at the plasma membrane and 15.0%±0.82 in the region corresponding to the membrane cytoskeleton (10–80nm); after stimulation with 0.5unit/ml thrombin for 2min this increased to 16.7%±0.22 and 40.4%±0.53, respectively.
DEVELOPMENTAL REGULATION OF GRANULE SIZE AND NUMBERS IN LARVAL SALIVARY GLANDS OF DROSOPHILA BY STEROID HORMONE ECDYSONE
Authors: Farkaš Robert , Šuáková Gabriela
Published: September 2002

The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well‐known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, ∼3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5×10−6m ecdysone.
LOCALIZATION OF β‐ENDORPHIN IN TETRAHYMENA BY CONFOCAL MICROSCOPY. INDUCTION OF THE PROLONGED PRODUCTION OF THE HORMONE BY HORMONAL IMPRINTING
Authors: Csaba G. , Kovács P.
Published: September 2002

The unicellular Tetrahymena has hormone receptors and hormones which are characteristic of higher vertebrates, as well as similar signal transduction pathways. In the present experiments, immunocytochemistry and confocal microscopy were used to study the presence and localization of β‐endorphin in Tetrahymena pyriformis GL. Endorphin (or endorphin‐like material) was localized in the cortical structures, oral field, cilia and nuclear envelope. One‐hour treatment with β‐endorphin (‘hormonal imprinting’) increased the presence of immunocytochemically demonstrable endorphin immediately and after 24h, and was especially strong after 96h of treatment. Simultaneous treatment with naloxone, an opioid antagonist, did not inhibit endorphin effect, but had an additive effect on endorphin production. Naloxone alone induced a very intensive accumulation of endorphin 96h after treatment. The results support the possibility of a hormone production being induced by the imprinting procedure, but the imprinter‐like effect of naloxone also points to the importance in this case of non‐discriminatory receptors also being involved in the process.
HELICAL EXTRUSIONS AT THE NUCLEAR ENVELOPE: POSSIBLE INVOLVEMENT IN NUCLEOCYTOPLASMIC TRAFFICKING
Authors: Wheatley Denys N.
Published: September 2002

The existence of intracytoplasmic helical inclusion 24h after serum stimulation of 3T3 cells which had previously been serum deprived (0.2%) for 2 days has been previously reported. Further evidence of their relationship with nuclear pores has been found, and their involvement in nucleocytoplasmic transport seems more probable. Helical formations resembling those originally seen have been found seemingly extruding from nuclear pores into the cytoplasm. However, it is unlikely that these structures contribute significantly to the translocation of material from the nucleus to the cytoplasm on a regular basis because their occurrence is rare. An association of the helical complexes with microfilaments has also been observed. Their relationship to annulate lamellae remain enigmatic.
THE EFFECTS OF EXHAUSTIVE EXERCISE ON THE ACTIVITY LEVELS OF CATALASE IN VARIOUS TISSUES OF MALE AND FEMALE RATS
Authors: Terblanche S. E.
Published: September 2002

The effects of exhaustive exercise on the activity levels of catalase (EC 1.11.1.6) in various tissues of male and female Sprague—Dawley rats (Rattus norvegicus) were investigated. Both the male and female rats were subdivided into an experimental group and a control group consisting of eight rats each. One group of each sex was subjected to a swimming session of 1h (experimental group) while the other group of each sex served as sedentary control groups. The tissues investigated were liver, heart, kidney and lung. The activity levels of catalase in all the tissues investigated were significantly (P< 0.05) elevated in both male and female rats as a result of exercise. The average increase in the activity levels of catalase in the various tissues investigated for both male and female rats was 417% (males 404%; females 430%). The male and female rats exhibited comparable activity levels of catalase in all the tissues investigated. The higher activity levels of catalase as a result of exercise might be indicative of a compensatory measure to counteract the possible detrimental effects associated with oxidative stress.
VARIABILITY IN E‐SELECTIN EXPRESSION, mRNA LEVELS AND sE‐SELECTIN RELEASE BETWEEN ENDOTHELIAL CELL LINES AND PRIMARY ENDOTHELIAL CELLS
Authors: Galley H.F , Blaylock M.G , Dubbels A.M , Webster N.R
Published: September 2002

Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E‐selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E‐selectin release. In cells exposed to tumour necrosis factor α (TNF‐α) and interleukin‐1β (IL‐1β), median (range) percentage of E‐selectin‐positive HUVECs increased from 1.6(0.9–6.2)% to 91.4(83.0–96.1)%, (P=0.001) using flow cytometry. In contrast, E‐selectin expression by ECV304 and EA.hy926 cell lines was 100‐fold lower. E‐selectin mRNA was detectable after 2h, maximal at 6h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF‐α/IL‐1β. sE‐selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E‐selectin, and may not be appropriate for studies of adhesion.
RESTING CYST WALL GLYCOPROTEINS ISOLATED FROM TWO COLPODID CILIATES ARE GLYCINE‐RICH PROTEINS
Authors: Izquierdo Adriana , Martín‐González Ana , Gutiérrez Juan Carlos
Published: September 2002

Two glycoproteins bands isolated from the cyst wall protein pattern of two colpodid ciliates,Colpoda inflata (gp46CI) and Colpoda cucullus (gp46CC) were analysed for their amino acid composition. Both glycoproteins are very rich in glycine and have a relatively high hydrophobicity, containing additionally many leucine and alanine residues. Their high degree of similarity is both quantitative and qualitative. Compared with just two previously published reports, their amino acid compositions are similar to those found in the hydrolysed cyst wall total proteins from the ciliatesC. steinii and Paraurostyla spp. The amino acid composition corroborates that they are indeed glycoproteins, because asparagine, an amino acid residue suitable for the attachment to N‐acetylglucosamine by its amide group (N‐glycan), is abundant in both proteins. We discuss our data in relation to other glycine‐rich proteins and a comparison with amino acid composition protein databases is carried out.
INVOLVEMENT OF THE PROTEIN KINASE PATHWAY IN MELANIN SYNTHESIS BY CHICK RETINAL PIGMENT EPITHELIAL CELLS
Authors: Kishi Hiroko , Mishima Hiromu K. , Yamashita Uki
Published: September 2002

Protein kinases are involved in a variety of cellular functions and cell proliferation in eyes. We have explored the involvement of protein kinase C (PKC) in cell proliferation and melanin synthesis by chick retinal pigment epithelial (RPE) cells in vitro. This was achieved by incubation of confluent RPE cells with known inhibitors of protein kinase, H‐7, W‐7, H‐8, and staurosporine. Chick RPE cells were cultured in the presence or absence of the protein kinase inhibitors for a 10‐day period. Effects of the inhibitors on cell proliferation and melanin synthesis, as an indication of cell differentiation, were assessed by counting the number of surviving cells and by measuring the melanin content in the cells, respectively. H‐7, W‐7, and staurosporine inhibited cell proliferation and increased melanin synthesis in a concentration‐dependent manner during culture; however, H‐8 did not produce these cellular effects. These findings indicate that PKC and calcium/calmodulin‐dependent kinase pathways are involved in the proliferation and differentiation of chick RPE cells.
CA2+MOBILIZATION IN THE HUMAN SUBMANDIBULAR DUCT CELL LINE A253
Authors: Andersson Charlotte , Zhang Ai‐Ling , Roomans Godfried M.
Published: September 2002

Ca2+mobilization induced by ATP, isoproterenol and the Ca2+‐ATPase inhibitor thapsigargin in the human submandibular duct cell line A253 was investigated using the Ca2+‐sensitive fluorescent indicator fura‐2. ATP and isoproterenol increased cytosolic free Ca2+([Ca2+]i) and subsequent exposure to thapsigargin after ATP or isoproterenol stimulation caused a further increase in [Ca2+]i. However, ATP and isoproterenol were not able to elicit a further increase in [Ca2+]iafter exposure of the cells to thapsigargin. Relatively few cells reacted to isoproterenol stimulation, but nearly all cells reacted to isoproterenol if ATP was added together with, or prior to isoproterenol stimulation. Moreover, the effect of ATP was potentiated by prior or simultaneous addition of isoproterenol. Furthermore, ATP decreased [Ca2+]iin the presence of thapsigargin probably due to agonist‐induced export of intracellular calcium. The results may suggest the existence of three thapsigargin sensitive pools; one opened by ATP acting through P2‐purinergic receptors and IP3, one opened by isoproterenol acting through β2‐adrenergic receptors, and a third pool not sensitive to ATP or isoproterenol.
GANGLIOSIDE‐MEDIATED METABOLIC SYNCHRONIZATION OF PROTEIN SYNTHESIS ACTIVITY IN CULTURED HEPATOCYTES
Authors: Brodsky Vsevolod Y. , Nechaeva Natalia V. , Zvezdina Natalia D. , Novikova Tatjana E. , Gvasava Inna G. , Fateeva Valentina I. , Prokazova Nina V. , Golovanova Natalia K.
Published: September 2002

An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3–0.5μm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06–0.2μm GM1 or 0.1–0.2μm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1‐antibodies for 24h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.
ULTRASTRUCTURE OF CHENOPODIUM RUBRUM L. APICES DURING THE EFFECT OF FUSICOCCIN AND PHOTOPERIODIC INDUCTION
Authors: Lozhnikova V. , Komarova E. , Muromtsev G.
Published: September 2002

Plant transition from vegetative to reproductive development is associated with ultrastructural changes in stem apices. Those seen in Chenopodium rubrum L. under the influence of fusicoccin in many ways resemble those induced by a short‐day treatment favourable to flowering. This suggests that fusicoccin can play a definite (physiological) role in plant development.
KINETIC CHARACTERIZATION OF CALCIUM UPTAKE BY THE RAT LIVER GOLGI APPARATUS
Authors: Rojas Patricio , Surroca Amarilys , Orellana Ariel , Wolff Daniel
Published: September 2002

We carried out a kinetic characterization of the Ca2+active transport in the rat liver Golgi Apparatus (GA) membrane. Calcium accumulation by vesicles of a GA enriched fraction was found to be a function of both Ca2+and ATP‐Mg concentrations, it was inhibited by 2μm thapsigargin but not stimulated by 3μm calmodulin. The kinetic parameter values obtained for the GA Ca2+pump were: Jmaxof 3.96nmol/mg min, Kmfor Ca2+of 0.150μm and two Km's for ATP of 1.14μm and 519μm. These results were almost identical to those obtained for the endoplasmic reticulum (ER) fraction, indicating that the GA Ca2+pump is a sarco/endoplasmic reticulum (SERCA) P‐type, analogous—if not identical—to that present in the ER.
APOPTOSIS IS A MODE OF CELL DEATH IN THE POLYKARYON‐FORMING UNIT ASSAY
Authors: Manti Lorenzo , Bowen Ifor D. , Mason Malcolm D. , Court Jon B.
Published: September 2002

In the polykaryon‐forming unit (PFU) assay, which defines cell survival as the ability to form a cytochalasin‐induced polykaryon of predetermined ploidy, the mode of PFU deletion is not known. Incubation of L5178Y‐S PFU in cytochalasin resulted in polyploidy (≥32C) and most polykaryons (>75%) ultimately underwent apoptosis, detected using chromatin condensation and externalised phosphatidylserine. However, large polykaryons carrying terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL)‐labelled DNA strand breaks were not observed, presumably due to rapid loss of DNA. Gamma irradiation of PFU prior to cytochalasin exposure caused a reduction in the frequency of highly polyploid cells (>16C), consistent with either a supra‐induction of apoptosis or a reduction in the ability of PFU to reach high ploidies. We conclude that L5178Y‐S PFU are deleted by apoptosis.
Cu—Zn SUPEROXIDE DISMUTASE INHIBITS LACTATE DEHYDROGENASE RELEASE AND PROTECTS AGAINST CELL DEATH IN MURINE FIBROBLASTS PRETREATED WITH ULTRAVIOLET RADIATION
Authors: Kimura Hirokazu , Minakami Hisanori , Otsuki Kunio , Shoji Akira
Published: September 2002

The effects of adding Cu—Zn superoxide dismutase (Cu—Zn SOD) to culture medium of the murine fibroblast cell line, L‐929, pretreated with UV‐B (312nm, 480mJ/cm2) have been investigated. Cell injury was monitored by the release of lactate dehydrogenase (LDH) into the medium, and cell death by the trypan blue exclusion test. UV‐B radiation induced cell death by apoptosis, as demonstrated by DNA fragmentation. Over the range 0.1–0.3μm Cu—Zn SOD, a significant dose‐dependent protection against cell death was obtained of the UV‐B exposed cells. Cell death correlated with the amount of LDH released into the medium, and Cu—Zn SOD treatment inhibited this. Heat‐denatured Cu—Zn SOD did not affect either cell viability or the release of LDH from the cells. Endogenous Cu—Zn SOD activity, monitored by chemiluminescence, decreased by 20% in UV‐B‐irradiated cells; the addition of 0.3μm exogenous Cu—Zn SOD to the medium did not affect intracellular Cu—Zn SOD activity. These results establish that Cu—Zn SOD added to extracellular medium can protect cells against injury caused by UV‐B exposure.
RADIAL SPINDLE AND THE PHENOTYPE OF THE MAIZE MEIOTIC MUTANT, DV
Authors: Shamina Natalia , Dorogova Natalia , Trunova Svetlana
Published: September 2002

The morphological phenotype of the maize meiotic mutant dv (divergent spindle) has been further analysed by visualization of the division spindle and examination of its fine structure in mother cells of pollen. Previous research showed that dv blocks convergence of spindle fibres at the poles. New observations reveal abnormalities caused by this mutation, with dv showing disturbances in nuclear envelope breakdown during vesiculation, preventing the spindle fibres from adopting a bipolar orientation (with convergence on the poles). The anomalies result in radial spindles which are similar to monoastral spindles in animal cells.
MODULATION OF THE RHYTHMIC PATTERNS OF EXPRESSION OF PHOSPHOPROTEIN PHOSPHATASES IN HUMAN LEUKAEMIA CELLS
Authors: Bhoola Rajesh , Hammond Kate
Published: September 2002

Temporal variations in the expression of phosphoprotein phosphatase 1 (PP1), phosphoprotein phosphatase 2A (PP2A) and protein tyrosine phosphatase 1B (PTP1B) were monitored in the human acute, promyelocytic leukaemia cell line, HL60. Granulocytic differentiation was induced using all‐trans retinoic acid (ATRA) and monocytic differentiation by phorbol‐12‐myristate‐13‐acetate (PMA). Expression of the enzyme proteins in cell extracts was determined by SDS‐PAGE and Western immunoblotting using specific antibodies. For PP1, a single immunospecific band of molecular mass 38kDa was detected corresponding to the catalytic subunit; induction of differentiation with either ATRA or PMA showed differences in the patterns of expression and, in the case of the latter, the mean value. Two immunospecific bands, of mass 34 and 37kDa, possibly corresponding to dephosphorylated and phosphorylated forms, respectively, were detected for PP2A, as well as a minor band of mass 46kDa; dynamic variations in the expression of all 3 forms were observed and there were differences between the control and treated cells. The catalytic domain of PTP1B was detected as a 46kDa band. A 42kDa form of the protein was also seen, which may represent a change in phosphorylation state, or be the result of proteolytic cleavage; usually the 46kDa band was the major form, but on occasion there was a change to predominance of the 42kDa band.
PROTEIN KINASE C IN ERYTHROLEUKAEMIA CELLS: TEMPORAL VARIATIONS IN THE EXPRESSION OF THE ALPHA, EPSILON AND ZETA ISOFORMS
Authors: Hammond K. D. , Savage N. , Littlewood M.
Published: September 2002

Determinations of mRNA and enzyme protein analyses for the protein kinase C (PKC) isoforms, alpha, epsilon and zeta, representing the classical, novel and atypical groups, respectively, in murine erythroleukaemic (MEL) cells revealed the occurrence of cyclic behaviour in expression. Such rhythmicity was not apparent when multiple analyses using only a single time‐point were considered; the importance of incorporating a time component into experimental design was demonstrated. On induction of differentiation over a period of five days with hexamethylene bisacetamide, changes in the patterns of expression of mRNA and protein were apparent, as compared with those of untreated, proliferating cells. Modulation of the dynamics of the various PKC isoforms may represent a regulatory mechanism for the control of a diversity of cellular functions.
MOSQUITO LARVAE PROTEINS MODULATE LUTEINIZING HORMONE AND PROLACTIN RELEASE IN PITUITARY CELLS OF NORMAL AND ESTROGENIZED RATS
Authors: Bolognani Federico , Goya Rodolfo Gustavo , Ronderos Jorge Rafael
Published: September 2002

Whilst looking for vertebrate growth factor homologues in insects, we found that a soluble fraction of a 12–80kDa molecular weight band peaking at 25kDa, isolated from mosquito larvae extracts by gel permeation chromatography, had a modulatory effect on mouse hepatocytes and adult human mononuclear cell proliferation. The effect disappeared after heating the extract at 90°C for 30min, suggesting that the active factor may be a protein. In order to determine the activity of the extract on cell function, we assessed the effect of the extract on pituitary hormone secretion in vitro. We assayed a dialyzed fraction (MW greater than 12kDa) of mosquito larvae for its effect on the release of luteinizing hormone (LH) and prolactin (PRL) from dispersed rat pituitary cells. In normal anterior pituitary (AP) cells we found that the extract had a stimulatory effect on LH release but an inhibitory action on prolactin secretion. In AP cells obtained from estrogen‐induced hyperplasia, the extract had an inhibitory effect on prolactin secretion. In all cases the effects were time‐ and dose‐dependent. Interference of the mosquito proteins with the radioimmunoassay was checked and found to be negligible. After a 60min incubation, cell viability was comparable in control and treated cells. Furthermore, the biological effect of the extract was thermally unstable. Our results suggest that mosquito larvae may share common factors with mammals, probably peptidic in nature, which are able to modulate cell function.