bluuebunny/crossref_metadata_2025 huggingface dataset:
GIANT VACUOLES OBSERVED IN DICTYOSTELIUM DISCOIDEUM
Authors: Yuan Aidong , Chia Catherine P.
Published: September 2002

Large intracellular vacuoles, >4μm in diameter and either round or oval‐shaped, were observed infrequently in Dictyostelium discoideum amoebae of axenically‐grown strain AX2 (only 1 in 106–108cells). These previously unreported single or multiple ‘giant’ vacuoles were more common, however, in newly germinated KAX3 cells (0.55% of the population) and AT‐Kneg, a strain that lacks an esterase (0.47% of the population). A vacuolar H+‐ATPase was enriched in their membranes of intracellular giant vacuoles, indicating that the vacuoles were related possibly to both endosomes and the contractile vacuole compartment. When monitored over time, giant vacuoles protruded from, and retracted back into cells under hyperosmotic conditions, suggesting an osmoregulatory role for these vacuoles. Some of the intracellular and protruded giant vacuoles harbored a fluid‐phase marker, fluorescein‐labeled dextran, implying a pinocytotic origin for the vacuoles.
CHANGES IN POLYAMINE METABOLISM DURING GLUCOCORTICOID‐INDUCED PROGRAMMED CELL DEATH IN MOUSE THYMUS
Authors: Hegardt Cecilia , Andersson Gunnar , Oredsson Stina M.
Published: September 2002

When mice are injected with dexamethasone, cortical thymocytes are deleted through programmed cell death (PCD). We have used this in vivo model system to investigate the kinetics of PCD and cell proliferation in relation to polyamine metabolism for 16h after injection of dexamethasone. As a marker for PCD, we used the appearance of a sub‐G1peak in the DNA histogram. When a sub‐G1peak appeared at 4h after dexamethasone treatment, the activity of the polyamine catabolic enzyme spermidine/spermine N1‐acetyltransferase (SSAT) was significantly increased and the activity of the polyamine biosynthetic enzyme S‐adenosylmethionine decarboxylase (AdoMetDC) was significantly decreased compared to the activities found in the thymi of control mice. Despite the significant changes in the activities of SSAT and AdoMetDC, the only change in the polyamine pool during the experimental period was that of putrescine. Presumably the complexity of this in vivo system masks changes in the spermidine and spermine pools that were expected in relation to the increased SSAT activity and decreased AdoMetDC activity.
EFFECTS OF THE MYOSIN INHIBITOR 2,3‐BUTANEDIONE MONOXIME (BDM) ON CELL SHAPE, LOCOMOTION AND FLUID PINOCYTOSIS IN HUMAN POLYMORPHONUCLEAR LEUCOCYTES
Authors: Urwyler N. , Eggli P. , Keller H. U.
Published: September 2002

We investigated the role of myosin in polymorphonuclear leucocyte (PMN) shape changes, locomotion, and fluid pinocytosis using the myosin inhibitor 2,3 butanedione monoxime (BDM). Treatment of resting spherical PMNs with BDM produced spheroid cells showing small continuous shape changes (IC50=15.5m m BDM) and occasionally small blebs. Cell polarity, as induced by the chemotactic peptide fNLPNTL or by colchicine, and locomotion were completely suppressed (IC50=8.4 to 10m m). Suppression of fNLPNTL‐ or colchicine‐induced cell polarity produced spheroid cells, suppression of PMA‐induced shape changes and fluid pinocytosis produced non‐motile spherical cells (IC50=25 to 30m m BDM). BDM suppressed formation of lamellipodia but not formation of blebs. Suppression of microvilli by BDM as observed in resting spherical cells was partially antagonized by PMA. The results suggest that myosin is involved in stabilizing the shape of resting spherical cells, including microvilli, and that myosin is required for cell polarity, locomotion, fluid pinocytosis and for formation of lamellipodia, but not for formation of blebs.
LOCAL REGULATION OF ANION SECRETION BY PITUITARY ADENYLATE CYCLASE‐ACTIVATING POLYPEPTIDE IN HUMAN COLONIC T84 CELLS
Authors: Leung P.S. , So S.C. , Lam S.Y. , Tsang L.L. , Chung Y.W. , Chan H.C.
Published: September 2002

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a novel hypothalamic peptide, which has been shown to exert various functions in a number of tissues, including exocrine and endocrine tissues. The present study investigated the role of local PACAP in the control of anion secretion by the human colonic T84 cell. Both bioactive forms of PACAP‐27 and PACAP‐38 gave rise to a dose‐dependent increase in the short‐circuit current (ISC). However, there was a reversal in the order of potency observed at different concentration ranges for the two bioactive forms. PACAP‐27 was greater than PACAP‐38 when the peptide concentrations were below 10n m; PACAP‐38 was greater than PACAP‐27 in the range of 10–80n m. The effects of both PACAP forms were restricted to the apical aspect of the T84 cell. The ISCresponses to both PACAP‐27 and PACAP‐38 were suppressed respectively by the non‐selective Cl−channel blocker, diphenylamine‐dicarboxylic acid (DPC), by the Ca2+dependent Cl−channel blocker, diisothiocyanatostilbene‐disulfonic acid (DIDS) and by the Ca2+chelator, BAPTA‐AM, indicating the involvement of Ca2+. The expression of PACAP was demonstrated and localized specifically to the perinuclear cytoplasm of the T84 cell using immunocytochemistry, indicating its epithelial origin. Thus, the present data suggest that, in addition to the well‐known cAMP‐dependent pathway, PACAP may play a role in regulating colonic Cl−secretion via a Ca2+‐dependent pathway, perhaps through two distinct PACAP receptor subtypes. Moreover, the regulation of anion secretion by T84 cells may be mediated by locally formed PACAP in an autocrine or paracrine fashion.
A NOVEL AND RAPID PREDICTION ASSAY FOR THE EFFECTIVENESS OF IL‐6 RECEPTOR SPECIFIC ANTISENSE OLIGONUCLEOTIDES BY PROLIFERATION INHIBITION OF AN INTERLEUKIN‐6 DEPENDENT CELL LINE
Authors: Varga Lia Valeria , Falus Andras , Pallinger Eva , Novák István , Fülöp Kristóf A. , Toth Sara , Rose‐John Stefan
Published: September 2002

Interleukin‐6 (IL‐6) belongs to a family of cytokines that use receptors consisting of a common signal‐transducing chain (gp130). Baf/3 cells transfected with the human IL‐6 receptor (IL‐6R) and gp130 (Baf/3‐gp130/IL‐6R) can only grow in medium containing IL‐6. We attempted to interrupt the signal transducing pathway of IL‐6 with the help of antisense oligonucleotides (ASOs) designed against the IL‐6R. We used 18 different kinds of antisense oligonucleotides of overlapping sequences around the translational start codon of the human IL‐6R. Sense ASOs were used as a control. The proliferation of cells was analysed by H‐thymidine incorporation. Cell surface expression of the IL‐6R was assessed by FACS analysis. We identified three ASOs which strongly inhibited the proliferation of IL‐6 dependent transfected Baf/3 cells. Flow cytometric studies on the suppression of surface expression of IL‐6R by ASOs showed a similar pattern. These results should help to clarify the structural requirements of functionally effective ASOs in the inhibition of IL‐6R.
TAXOL INDUCED BCL‐2 PROTEIN PHOSPHORYLATION IN HUMAN HEPATOCELLULAR CARCINOMA QGY‐7703 CELL LINE
Authors: Cheng Samuel C.‐S. , Luo Dan , Xie Yong
Published: September 2002

Bcl‐2 family proteins play a critical role in the regulation of apoptosis. Treatment of a human hepatocellular carcinoma cell line, QGY‐7703, with Taxol induced apoptosis and Bcl‐2 protein phosphorylation. Microscopic observation indicated that apoptotic bodies (0–15%) of Taxol‐treated QGY cells appeared after 12h of treatment, and apoptotic QGY cells gradually increased to 40% after 24h and 70% after 48h. A DNA fragmentation assay showed that Taxol induced genomic DNA cleavage into 200bp DNA fragments. Bcl‐2 protein was phosphorylated in Taxol‐treated QGY cells within 3h of treatment, and continued gradually up to 24h. By 48h, the protein was unphosphorylated. Other Bcl‐2 family proteins, including Bax (a heterodimerization partner of Bcl‐2), Bcl‐XL, Bak and Bad, were expressed, but at constant levels. The results show a close correlation between Bcl‐2 phosphorylation and apoptosis in QGY cells. The inactivation of Bcl‐2 protein phosphorylation could be one of the key mechanisms needed for the induction of apoptosis in Taxol‐treated QGY cells.
RENAL TUBULAR CELL NECROSIS AND APOPTOSIS IN TRANSPLANTED KIDNEYS
Authors: Toronyi E. , Lord R. , Bowen I. D. , Perner F. , Szende B.
Published: September 2002

Renal wedge biopsies were taken from donors' kidneys immediately at the end of cold ischaemia and 30min after transplantation in 11 cases. Five renal grafts showed immediate and six showed delayed renal function clinically. The ratio of apoptotic and necrotic renal tubular cells and Ki67 activity was determined in both biopsies. Necrotic and apoptotic as well as proliferating renal tubular cells were seen in all samples. Both apoptotic and proliferative activity was decreased in samples taken 30min after transplantation in cases of immediate renal function compared to the samples taken before transplantation. This phenomenon was not observed in cases of delayed renal function.
PDGF‐BB AND IGF‐I USE DIFFERENT SIGNALING PATHWAYS TO INDUCE FIBRONECTIN SYNTHESIS IN CULTURED RAT THORACIC AORTIC SMOOTH MUSCLE CELLS
Authors: Lo Chu S.
Published: September 2002

Fibronectin, an extracellular matrix protein, acts as an early signal in initiating cell proliferation. Results have indicated that platelet‐derived growth factor BB (PDGF‐BB) and insulin‐like growth factor‐I (IGF‐I) both enhance fibronectin gene expression. Genistein inhibits PDGF‐BB‐induced fibronectin levels without inhibiting IGF‐I‐induced fibronectin levels. It indicates that PDGF‐BB and IGF‐I utilize separate signaling pathways to induce the synthesis of fibronectin.
AUTOCRINE‐ACTING EARLY SECRETED MITOGENIC ACTIVITY: PRODUCTION AND RESPONSIVENESS IN CULTURES OF NORMAL HUMAN KERATINOCYTES AS A FUNCTION OF IN VIVO AND IN VITRO AGE
Authors: Tenchini Maria Luisa , Savant Fabio , Paini Cinzia , Montefusco Maria Claudia , Donati Vera , Malcovati Massimo
Published: September 2002

Cultured epithelial grafts are used in the clinical treatment of both non‐healing and acute partial‐thickness wounds, owing to their ability to stimulate endogenous re‐epithelialization. We have previously demonstrated that during the first 24h following plating, human epidermal keratinocytes secrete an autocrine‐acting mitogenic activity. Since the biological activity of cultured grafts is believed to decrease with cellular age, the effect of both in vivo andin vitro keratinocyte age on the secretion of this mitogenic activity, as well as on responsiveness to this activity, was studied. Keratinocytes from donors ranging in age from 2 to 81 years were analysed at increasing in vitro population doublings. Secretion into the medium of the mitogenic activity was not affected by either in vivo or in vitro cellular ageing, while responsiveness of keratinocytes to this mitogenic activity was age‐related. These results suggest that cultured grafts from elderly donors may be effective in wound treatment.
1α, 25‐DIHYDROXYVITAMIN D3 SUPPRESSES THE EFFECT OF STREPTOZOTOCIN‐INDUCED DIABETES DURING CHEMICAL RAT LIVER CARCINOGENESIS
Authors: Saha Barun Kanti , Sarkar Alok , Basak Ranjan , Chatterjee Malay
Published: September 2002

The effect of streptozotocin‐induced diabetes in male Sprague—Dawley rats was investigated to ascertain whether it has had any modulating role in hepatocarcinogenesis. Hepatocarcinogenesis was initiated with a single sub‐necrogenic dose of diethylnitrosamine (DEN) (125mg/kg body weight, i.p.) whilst acute diabetes was produced with a single i.p. injection of streptozotocin (STZ) (65mg/kg body weight). STZ was administered either before or after initiation with DEN at 3‐week intervals. With this basic experimental regimen, the effect of an antioxidant vitamin, 1α, 25‐dihydroxyvitamin D3(VD) (0.3μg/ 0.1ml propylene glycol per os twice a week), was investigated with effect from 4 weeks prior to the exposure of DEN or STZ. Primary routine histopathology, hepatic nodular morphometric analysis and major preneoplastic antioxidant and drug metabolising enzymes were tested either with or without VD treatment in different experimental and control groups. Observation of the hepatic nodulogenesis, pathology and level of the antioxidant and drug metabolising enzyme pattern of the tissue showed a marked protection in different experimental groups of rats treated with VD. It may be that VD could elicit an anticarcinogenic potential in the aforesaid regimen by resetting the effects of these biomarkers induced by DEN and/or STZ. We further propose that STZ, when administered 3 weeks after DEN, caused massive damage where its action in vivo could be comparable with any known promoter that could propel the process of carcinogenesis more efficiently than when it was applied before the carcinogen.
KCl STIMULATION AND ULTRASTRUCTURAL RESPONSES OF TANNIC ACID‐STAINED CHOLINERGIC SYNAPTIC TERMINALS
Authors: Fox Geoffrey Q. , Kriebel Mahlon E.
Published: September 2002

The quantal‐vesicular hypothesis equates miniature end‐plate potentials (MEPPs) with fusions of synaptic vesicles. MEPP production thus predicts vesicle losses, increases in vesicle fusions and increases in terminal plasma membrane. MEPP production and these ultrastructural parameters have been evaluated in the cholinergic presynaptic terminals of skate electric organ following tannic acid saline incubation, known to promote capture and selective staining of dense‐core granule fusions, and KCl stimulation, known to elevate MEPP production dramatically in these cholinergic terminals. After pretreatment in tannic acid—elasmobranch saline, KCl stimulation produced MEPPs at 40/s/μm2of terminal surface for several minutes with gradual reduction to spontaneous levels by 25–30min. No loss of vesicles, no vesicle fusions, no expansions of plasma membrane and no tannic acid enhanced staining of vesicles or vacuoles accompanied the generation of 800MEPPs/μm3of terminals having densities of 567 vesicles/μm3. No ultrastructural footprints were found to support the notion that unnaturally high rates of vesicular exocytosis had occurred.
FUSION OF SENDAI VIRUS WITH VESICLES OF OLIGOMERIZABLE LIPIDS: A MICROCALORIMETRIC ANALYSIS OF MEMBRANE FUSION
Authors: Ravoo Bart Jan , Weringa Wilke D. , Engberts Jan B. F. N.
Published: September 2002

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2 ‐di‐n ‐hexadecyloxypropyl‐ 4 ‐(beta‐nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37°C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively‐charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy‐driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein‐rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se.
INHIBITION OF FOCAL ADHESION KINASE EXPRESSION CORRELATES WITH CHANGES IN THE CYTOSKELETON BUT NOT APOPTOSIS IN PRIMARY CULTURES OF CHICK EMBRYO CELLS
Authors: Ridyard Marc S. , Sanders Esmond J.
Published: September 2002

Cell adhesion to the extracellular matrix through integrin receptors can activate signaling cascades within the cell. Focal adhesion kinase (FAK) is a protein tyrosine kinase activated by integrin adhesion. The role of FAK within the cell is not clear, although evidence suggests roles in cell motility or the regulation of adhesion‐dependent cell survival. We have treated primary cultures of chick embryo cells with antisense oligonucleotides to FAK to reduce the level of FAK protein expression. Levels of the related protein, proline‐rich tyrosine kinase 2 (Pyk2) and the FAK substrate paxillin, were unaffected by the addition of oligonucleotides, whereas FAK expression was reduced by 70%. Levels of apoptotic cell death did not significantly increase after the addition of oligonucleotides. However, there was a change in the distribution of focal adhesion sites from a uniformly distributed pattern to a mainly peripheral pattern. This was accompanied by a loss of stress fibers and an increase in the peripheral actin cytoskeleton, as the cells became rounded. These results suggest that in these early embryonic cells, FAK expression regulates the arrangement of focal adhesions and the cytoskeleton that result in a motile phenotype, but that FAK does not appear to regulate apoptosis.
CELLULAR DISTRIBUTIONS OF THE ERM PROTEINS IN MDCK EPITHELIAL CELLS: REGULATION BY GROWTH AND CYTOSKELETAL INTEGRITY
Authors: Woodward Anna M. , Crouch Dorothy H.
Published: September 2002

The highly homologous ERM (ezrin/radixin/moesin) proteins, molecular cross‐linkers which connect the cell membrane with the underlying cytoskeleton, have molecular weights of 81, 80 and 78kDa respectively. We present data which shows significant variation in the molecular weight and presence of multiple forms of ERM proteins in different cell lines, such that specific antibodies to each protein are essential for unambiguous detection. Biochemical fractionation of MDCK cells demonstrates that although the individual ERM fractionation patterns are unaltered by cell density, the multiple forms of moesin each associate with different subcellular fractions. Since ERM proteins can exist in dormant or active conformations corresponding to their phosphorylation state, we propose that the partitioning of ERM proteins between subcellular compartments may depend on their activation status. In addition, we show that when the co‐localization between ezrin and F‐actin is disrupted by cytochalasin D, MDCK cells undergo a dramatic morphology change during which long, branching, ezrin‐rich protrusions are formed. Consistent with other workers, our data suggest that maintenance of ezrin: F‐actin interactions are required for the maintenance of normal cellular morphology.
LAMININ‐2/4 FROM HUMAN PLACENTA IS A BETTER ADHESION AGENT FOR PRIMARY KERATINOCYTES THAN LAMININ‐1 FROM EHS SARCOMA
Authors: Gorelik J. V. , Cherepanova O. A. , Voronkina I. V. , Diakonov I. A. , Blinova M. I. , Pinaev G. P.
Published: September 2002

A comparison of the adhesion of human primary keratinocytes to laminin‐1 from murine EHS sarcoma and laminin‐2/4 from human placenta was carried out using two methods, cell adhesion to substrates covered with the laminin isoforms, and interaction of keratinocytes from suspension with latex beads coated with the proteins. Laminin‐2/4 was considerably more potent as a promoter of attachment of primary human keratinocytes than laminin‐1 (and fibronectin), with increased attachment of cells correlating well with the number of latex bead binding sites. Only small cells of diameter of less than 20μm bound more than 5 beads. Staining of keratinocytes with involucrin antibodies confirmed the existence of an inverse relationship between laminin‐2/4‐coated bead binding and differentiation.
PARTICIPATION OF THE Ca2+‐CALMODULIN‐ACTIVATED KINASE II IN THE CONTROL OF METAPHASE‐ANAPHASE TRANSITION IN HUMAN CELLS
Authors: Petzelt Christian P. , Kodirov Sodikdjon , Taschenberger Grit , Kox Wolfgang J.
Published: September 2002

Treatment of HeLa S3 cells growing in suspension, and of endothelial cells ECV 304 growing as a monolayer, with the inhibitor of the Ca2+‐calmodulin activated Kinase II KN‐93, blocks cells at metaphase for 15min (HeLa cells) and 30min (ECV cells). Thereafter cells resume mitosis and enter anaphase. The inactive isomer KN‐92 does not show such effects. The results show the involvement of the CaM K II system in the regulation of the metaphase‐anaphase transition whereby the activation of the Kinase II, in particular by calmodulin appears to be affected, the residual autophosphorylation of the CaM K II system apparently sufficing after 15 to 30min to release the cells into anaphase. The results are compared with the metaphase‐blocking effects of the noble gas xenon, where the xenon‐induced block can be overcome by small intracellular increases of Ca2+, thus indicating the CaM K II system as a possible target for xenon.
CYTOCHROME‐C LOCALIZES IN SECRETORY GRANULES IN PANCREAS AND ANTERIOR PITUITARY
Authors: Soltys Bohdan J. , Andrews David W. , Jemmerson Ronald , Gupta Radhey S.
Published: September 2002

We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome‐c in normal rat tissues, employing a wide variety of monoclonal and polyclonal antibodies against mammalian cytochrome‐c. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows highly specific labeling of mitochondria in all tissues examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen and thyroid. In pancreatic acinar cells and anterior pituitary, however, there was also strong cytochrome‐c reactivity in zymogen granules and growth hormone granules, respectively. In the pancreas, strong immunoreactivity is also detected in condensing vacuoles and in the acinar lumen. Immunocytochemical controls included (i) use of monoclonal antibodies to horse cytochrome‐c which recognize an epitope not present in rat cytochrome‐c, (ii) preadsorption of antibodies with purified cytochrome‐c, and (iii) omission of the primary antibody. The indicated presence of cytochrome‐c outside mitochondria in certain tissues under normal physiological conditions raises interesting questions concerning translocation mechanisms and the cellular functions of cytochrome‐c.
RESTORATION OF THE RAT UROTHELIUM AFTER CYCLOPHOSPHAMIDE TREATMENT
Authors: Romih Rok , Koprivec Dasa , Martincic Draga Stiblar , Jezernik Kristijan
Published: September 2002

Processes leading to the recovery of a normal three‐layered urothelium from a hyperplastic urothelium induced by cyclophosphamide (CP) treatment in rats have been investigated. A single intraperitoneal (ip) dose of CP caused extensive loss of cells from urothelium, but the remaining cells started to express epidermal growth factor receptor (EGFR) in their plasma membranes. On day 2 after CP injection, proliferating cell nuclear antigen (PCNA) immunohistochemistry showed a rapid increase in positively stained nuclei, from which a hyperplastic urothelium developed, composed of undifferentiated cells expressing EGFR over the entire plasma membrane. Subsequently, EGFR gradually disappeared from the apical plasma membrane but remained in the basolateral membranes. After day 6, PCNA‐positive nuclei in all cell layers decreased, except in basal cells. Apoptotic cells were detectable by the TUNEL assay at day 2, and increased in number in all layers of the hyperplastic urothelium until day 10, returning to the control levels by day 14. Electron microscopic evidence showed that apoptotic cells were either pinched off into the bladder lumen or phagocytosed by the neighbouring urothelial cells. Thus, the urothelium responds to the damage by intense proliferation for a week, resulting in an undifferentiated hyperplastic state. Differentiation of superficial cells then begins and damaged cells are gradually removed by apoptosis until the three‐layered urothelium is fully restored by two weeks following CP treatment.
EARLY DEVELOPMENT OF THE GUT: NEW LIGHT ON AN OLD HYPOTHESIS
Authors: Rawdon Benjamin B.
Published: September 2002

This review deals with the early development of the gut. It draws largely on information provided from the study of avian embryos. Evidence that concerns the early determination of the regional fate of the endoderm and mesoderm of the gut is reviewed. Gut endoderm can undergo a limited degree of differentiation from a remarkably early age when cultured in the absence of mesoderm and there is evidence that points to the establishment of a pre‐pattern in the early mesoderm before the genes responsible for patterning in gut are active. Initially, at least at cranial levels, those parts of the mesoderm and endoderm that are in contact are not those parts that will ultimately be in apposition; the consequence of this for any signalling between these layers is considered. In the light of the above information, the probable role of mesenchyme in gut development is re‐examined.
REGULATION OF GENE EXPRESSION BY GC‐RICH DNA CIS ‐ELEMENTS
Authors: Hapgood Janet P. , Riedemann Johann , Scherer Sonja D.
Published: September 2002

GC‐rich DNA cis elements are important transcriptional regulatory elements present in the promoter, enhancer and locus control regions of many eukaryotic genes from several species. This review attempts to examine the structure, function and biological significance of GC‐rich cis ‐regulatory elements and their cognate binding proteins, with a view to understanding their role in regulation of gene expression.
ASSESSMENT OF THE ANTITUMOUR ACTIVITY OF TARGETED IMMUNOSPECIFIC ALBUMIN MICROSPHERES LOADED WITH CISPLATIN AND 5‐FLUOROURACIL: TOXICITY AGAINST A RODENT OVARIAN CARCINOMA IN VITRO
Authors: Truter Ernest J. , Santos Aldina S. , Els Willem J.
Published: September 2002

The 3‐(4,5 dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide (MTT) assay is used successfully to estimate the number of viable cells in drug screening trials. We used the MTT assay to assess the viability of a rodent ovarian carcinoma cell line (DMBA‐OC‐1R) after exposure to combinations of cisplatin and 5‐fluorouracil as free drug and in encapsulated (conjugated and unconjugated) forms. After 48h of exposure to free drugs, a significant trend towards cell cytotoxicity could be observed and this was well established by 120h. Cells treated with drug‐containing immuno‐microspheres showed a similar initial decrease in cell viability after 96h, and this was maintained for 128h. These results suggest that immuno‐microspheres loaded with chemotherapeutic drugs have the potential to be successfully used in the treatment of ovarian cancer.
THE EFFECTS OF HYDROSTATIC PRESSURE‐INDUCED CHANGES ON THE CYTOSKELETON AND ON THE REGULATION OF GENE EXPRESSION IN PHEOCHROMOCYTOMA (PC‐12) CELLS
Authors: Wilson Ronald G. , Zimmerman Selma , Zimmerman Arthur M.
Published: September 2002

The purpose of this investigation was to determine the relationship of hydrostatic pressure‐induced changes in the cytoarchitecture to regulation of gene expression in PC‐12 cells. Hydrostatic pressure disrupts the cytoskeleton, decreases tubulin and actin mRNA levels and causes changes in the localization of tubulin and actin mRNA. Actin mRNA levels, at 6000 and 10,000psi for 20min, were reduced to 78% and 64%, respectively, in undifferentiated cells and to 81% and 72%, respectively, in 4‐day differentiating cells, relative to untreated controls. Tubulin mRNA levels, at 6000 and 10,000psi for 20min, were reduced to 75% and 67%, respectively, in undifferentiated cells and to 84% and 74%, respectively, in 4‐day differentiating cells. Changes in the localization of mRNA in the soluble and cytoskeletal fractions were determined by measuring the pressure level where the mRNA level in the cytoskeletal fraction equals the mRNA level in the soluble fraction. This measurement was designated the cytoskeletal/soluble fraction index (CSFI50). CSFI50measurements indicated that following hydrostatic pressure, actin mRNA cytoskeletal association was more stable than tubulin mRNA cytoskeletal association. The addition of chemicals which stabilize or destabilize microtubules and microfilaments to pressure treatment resulted in additional changes in the CSFI50.
IMPLICATION OF ANNEXIN 1 IN PHAGOCYTOSIS: EFFECTS OF N‐TERMINAL DOMAIN DELETIONS AND POINT MUTATIONS OF THE PHOSPHORYLATION SITE SER‐27
Authors: Kusumawati Asmarani , Liautard Jean‐Pierre , Widada Sri Joannes
Published: September 2002

Directed mutagenesis, in the form of deletions and point mutations, was used to investigate the regulatory importance of the N‐terminal domain of annexin 1. Wild‐type and mutant forms were fused to green fluorescent protein (GFP) to track their localization and introduced in to J‐774A.1 cells by transfection. The fusion of annexin 1 to GFP at the N‐ or C‐terminal end did not alter the cellular distribution or co‐localization with phagosomes. The effects of mutations were determined according to these characteristics. The prominent effect resulted from S27E mutation which mimics the phosphorylated state of Ser‐27. Although still retaining the granular structures in the cytoplasm, S27E annexin 1 failed to associate with the phagosomal protein complex. This suggests an essential regulatory role of the phosphorylation of residue 27 in annexin 1 function.
ERYTHROCYTE SODIUM/HYDROGEN EXCHANGE INHIBITION BY (−) EPICATECHIN
Authors: Matteucci Elena , Rizvi Syed Ibrahim , Giampietro Ottavio
Published: September 2002

Epicatechin, a flavonoid belonging to the group of compounds collectively called catechins, have been reported to possess insulin‐like properties. Besides their anti‐diabetic properties, catechins also show growth inhibition. Since cytosolic pH (pHi) plays a role in cell proliferation and the Na/H exchanger (NHE) is the major pH (pHi) regulatory mechanism, we undertook in vitro studies with human erythrocytes to examine the effect of (−) epicatechin (EC) on the NHE1 isoform. NHE activity was measured in eight healthy volunteers, eight type 1 diabetics, and nine type 2 diabetics, following 30min incubations at 37°C with either 1m m epicatechin, 10−9m insulin or solvent alone. NHE activity was elevated in both groups of patients (P< 0.05). Epicatechin caused a 93% decrease in Na/H antiport activity in health controls, 89 and 86% in type 1 and type 2 diabetics, respectively (P< 0.001). Insulin caused a 36% decrease in antiport activity only in the type 2 diabetic group (P< 0.05). The strong inhibition of erythrocyte NHE1 (the ubiquitously present isoform) by epicatechin may have important implications. NHE1 inhibition could be one of the major mechanisms underlying the antiproliferative effects of catechins.
APPLICATION OF CELL ENGINEERING TECHNOLOGY TO THE TUMOUR IMMUNOTHERAPEUTIC DRUG: A REVIEW
Authors: Chen Z. N. , Xing J. L. , Bian H. J. , Mi L. , Jiang J. L.
Published: September 2002

The concept of cell engineering is frequently mentioned in recent years. It's a comprehensive technology which is very powerful and useful. By using it, scientists can produce many kinds of proteins and other biological moleculars in large scale that are in great demand for the human being. Among these products, most can be used for tumor immunotherapy and have been confirmed to be very effective in tumour control or subsidiary treatment. Of them, most common and useful drugs include three sorts; tumour vaccines, cell factors and monoclonal antibodies. The paper will discuss in detail related drugs application advances in tumour immunotherapy.
SUPPRESSION OF ATP‐INDUCED CL−SECRETION BY ENHANCED EXPRESSION OF EPITHELIAL NA+CHANNELS IN MOUSE ENDOMETRIAL EPITHELIUM
Authors: Wang X. F. , Tsang L. L. , So S. C. , Chan H. C.
Published: September 2002

We have studied the effect of enhanced expression of epithelial Na+channels (ENaC) on the ATP‐induced Cl−secretion in the mouse epithelium using short‐circuit current (ISC) and RT‐PCR techniques. The amiloride sensitivity of basal current (Ib) across the cultured endometrial epithelia was found to vary with the magnitude of the Ib, the higher the Ibthe greater its sensitivity to amiloride, indicating possible elevation of ENaC. However, the magnitude of ATP‐induced ISC, previously demonstrated to be mediated by Ca2+‐activated chloride channel (CaCC), decreased as the amiloride sensitivity of the Ibincreased, suggesting a possible inhibitory effect of elevated expression of ENaC on ATP‐mediated chloride secretion. The Matrigel treatment for culturing the endometrial epithelia affected the amiloride sensitivity of the Ibas well as the ATP‐induced ISCreversedly. Competitive RT‐PCR demonstrated that the expression of both ENaC γ subunits and CaCC was enhanced in Matrigel‐treated cultures. However, the observed reduction in the ATP‐induced or CaCC‐mediated ISCcould not be explained by the CaCC expression pattern. These data suggest that inhibition of CaCC function is due to enhanced ENaC expression. Therefore, in addition to interacting with CFTR, ENaC also appears to interact with CaCC in the mouse endometrial epithelium. Physiologically the present findings indicate that enhanced expression of ENaC leads to suppression of other Cl−channels, such as CFTR and CaCC, thereby preconditioning the endometrium in favour of overall salt and water absorption as observed during embryo implantation.
EFFECT OF HYALURONAN ON MMP EXPRESSION AND ACTIVATION
Authors: Isnard N. , Legeais J‐M. , Renard G. , Robert L.
Published: September 2002

Matrix metalloproteases (MMPs) play a crucial role in tissue remodelling in a variety of physiological and pathological processes. Hyaluronan is also involved in the same processes. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan and also of MMPs. The activity of MMPs has been shown to be regulated at the level of transcription and activation of the zymogen form.

In order to explore the possible relationship between matrix components and especially hyaluronan, we studied the effect of hyaluronan on MMP expression (biosynthesis and activation) in the culture of human skin fibroblasts and corneal keratocytes (explant cultures and cell cultures). These cells were shown to exhibit distinct phenotypes as far as matrix biosynthesis is concerned. Using a synthetic substrate N‐Suc(ala)3pNA, we measured elastase‐type endopeptidase activity produced by fibroblasts and keratocytes and characterized the MMPs by zymography.

Hyaluronan added to fibroblast cultures stimulated the membrane‐bound elastase‐type endopeptidase activity in a dose dependent fashion. Similar results were obtained with keratocyte cultures. In the presence of 1mg/ml hyaluronan there was an increase in MMP expression and also an activation of latent MMPs both by fibroblasts and keratocytes.
GP130‐SPECIFIC ANTISENSE OLIGONUCLEOTIDES INHIBIT IL‐6 SIGNAL INDUCING JUNB MRNA TRANSCRIPTION IN THE HUMAN HEPATOMA CELL LINE, HEPG2
Authors: Varga Valéria Lia , Fülöp András Kristóf , Holub Marianna Csilla , Tóth Sára , Szalai Csaba , Falus András
Published: September 2002

The biosynthesis of interleukin‐6 receptor (IL‐6R) and gp130 in vitro was blocked using specific antisense oligonucleotides (ASO) in HepG2 liver cells and the efficacy of various ASOs was tested on the generation of IL‐6‐induced junB mRNA. We used three ASOs specific for the IL‐6 receptor, three specific for gp130 and a control (nonsense) oligonucleotide specific for ∍‐chain of IgE (not expressing in HepG2 cells). Our data indicate that a gp130‐specific ASO,g2, was the most effective blocker of IL‐6‐induced junB mRNA, whilst the IL‐6 receptor ASOs alone were ineffective. The mechanism of gene inactivation by ASO treatment was partially elucidated by demonstration of the loss of gp130 mRNA from cells treated with ASOs showing functional efficacy. Our data may help to design antisense oligonucleotides that are effective in therapy (e.g. as anti‐inflammatory agents) in the future.
RELEASE OF IL‐4 AND IL‐5 FROM HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS CO‐CULTURED WITH JAPANESE CEDAR POLLEN ANTIGENIN VITRO
Authors: Kimura Hirokazu , Shibata Yukino , Minakami Hisanori , Hashizume Setsuko , Oba Hiromi , Sakaguchi Masahiro , Otsuki Kunio , Kozawa Kunihisa , Kato Masahiko
Published: September 2002

The Japanese cedar pollen (JCP) is a major allergen with respect to pollinosis in Japan. It is believed that interleukin‐4 (IL‐4) and interleukin‐5 (IL‐5) derived from lymphocytes and other cells play a pivotal role in allergic reactions. We investigated whether the JCP antigen stimulates the release of these cytokines by peripheral blood mononuclear cells (PBMCs). PBMCs from eight adults (five adults with JCP pollinosis and three adults without JCP pollinosis) were co‐incubated with purified JCP antigens. IL‐4 was released in response to JCP antigens in six of the eight subjects at 24h and in three subjects at 48h. IL‐4 release at 24h occurred in all five subjects with JCP pollinosis but in only one of the three subjects without pollinosis. IL‐5 was released in response to the JCP antigen in five of the eight subjects at 24h and 48h, including four of the five subjects with JCP pollinosis and one of the three subjects without pollinosis. These results suggest that PBMCs were more likely to release IL‐4 and IL‐5 in the presence of JCP pollinosis.
DETECTION OF SUBMICROSCOPIC MAGNETITE PARTICLES USING REFLECTANCE MODE CONFOCAL LASER SCANNING MICROSCOPY
Authors: Green C. R. , Holloway H. , Walker M. M.
Published: September 2002

Light microscopy and transmission electron microscopy work at such different scales that some components of cells may be too small to detect using light microscopy but too dispersed among cells within tissues to be discovered using electron microscopy. We have used reflectance mode confocal laser scanning microscopy to detect single‐domain magnetite crystals in both live and resin‐embedded preparations of magnetotactic bacteria. We show that reflections from bacterial cells are uniquely associated with the magnetite, which underpins the magnetotactic response of the bacteria. En bloc viewing shows that relatively large volumes of material can be searched with sufficient resolution to enable detection of submicroscopic particles. The techniques reported here may be of interest to others wishing to detect submicroscopic objects dispersed in large volumes of tissue.
EFFECT OF VASOACTIVE PEPTIDES ON TETRAHYMENA. CHEMOTACTIC PROPERTIES OF ENDOTHELINS (ET‐1, ET‐2, ET‐3, FRAGMENT 11–21 OF ET‐1 AND BIG ENDOTHELIN‐1): A SHORT‐TERM INDUCIBLE SIGNALLING MECHANISM OF CHEMOTAXIS
Authors: Kohidai László , Tóth Katalin , Ruskoaho Heikki , Csaba György
Published: September 2002

The unicellular Tetrahymena is a sensitive model for the study of chemotaxis induced by endothelins. In short‐term chemotactic responses, ET‐2 and ET‐3 were chemorepellent, compared to the referent control (culture medium) and chemoattractant, ET‐1. These differences suggest that the change of some aromatic residues in the loop region (residues 5–9) of the ET‐1 abrogates its chemoattractant character. The response of Tetrahymena is highly selective, since substitution of two amino acids are enough to cause this alteration in (behavioural) response. Such a change seems to be more important than the loss of the entire first 10 amino acids (in ET‐1 fragment 11–21), since after this it acquires some chemoattractive effect of ET‐1. Selection with ET‐3 rigorously stimulated the cell's responsiveness to the medium, this ability was abolished by the repeated ET‐3 treatment. Big endothelin‐1 was repellent in all concentrations. These experiments demonstrate the very sensitive discriminating capacity of chemotactic responsiveness at a low level of phylogeny. Chemotactic selection with endothelins underlines the possibility that there may be separate mechanisms responsible for the short‐term chemotactic responses and the long‐lasting effects of chemotactic selection.
HEAT SHOCK RESPONSE AND AGEING: MECHANISMS AND APPLICATIONS
Authors: Verbeke Philippe , Fonager Jannik , Clark Brian F. C. , Rattan Suresh I. S.
Published: September 2002

Ageing is associated with a decrease in the ability of cells to cope with environmental challenges. This is due partly to the attenuation of a primordial stress response, the so‐called heat shock (HS) response, which induces the expression of heat shock proteins (HSPs), composed of chaperones and proteases. The attenuation of the HS response during ageing may be responsible for the accumulation of damaged proteins as well as abnormal regulation of cell death. Maintenance of the HS response by repeated mild heat stress causes anti‐ageing hormetic effects on cells and organisms. Here, we describe the molecular mechanism and the state of the HS response as well as the role of specific HSPs during ageing, and discuss the possibility of hormetic modulation of ageing and longevity by repeated mild stress.
BASAL LEVELS OF METALLOTHIONEIN I AND II EXPRESSION IN MOUSE EMBRYO FIBROBLASTS ENHANCE GROWTH IN LOW FOLATE THROUGH A CELL CYCLE MEDIATED PATHWAY
Authors: Zhu Wei‐Yong , Melera Peter W.
Published: September 2002

The impact of basal (non‐induced) expression levels of metallothionein I and II on the growth of mouse embryo fibroblasts in standard DMEM/F‐12 containing 8.8μm folic acid, and in DMEM/F12 without hypoxanthine, thymidine or folic acid, containing 15n m or 15p m[6S]‐folinic acid, was assessed by comparing wild‐type MT (+/+) and homozygous null MT (−/−) cell lines. No difference in growth rate was observed between the two in DMEM/F12, although MT (−/−) cells displayed a 6‐fold decrease in p27Kip1, a two fold increase in p53 and a slight increase in p21Waf1. After 6 days in culture, the growth rate for MT (−/−) cells in 15n m or 15p m[6S]‐folinic acid was half that of MT (+/+). After an additional 6 days in 15n m folate, both MT (+/+) and (−/−) cells maintained their respective growth rates, while those in 15p m had ceased to grow. During the initial 6 days in 15n m folate, neither cell population displayed an increase in apoptosis or a change in cell cycle distribution, even though MT (−/−) cells sustained an additional 4‐fold increase in p21Waf1and a 6‐fold decrease in cyclin E expression. At day 12, however, the MT (−/−) population, but not MT (+/+), underwent a 7‐fold increase in apoptosis coupled with a 3 fold increase in S phase cells. Hence, the basal level of MT I and II constitutively expressed in MT (+/+) cells enhances growth in 15nM [6S]‐folinic acid by preventing S phase arrest and apoptosis.
CYCLIN B1 PROMOTER ACTIVITY AND FUNCTIONAL CDK1 COMPLEX FORMATION IN G1 PHASE OF HUMAN BREAST CANCER CELLS
Authors: Barrett Katherine L. B. , Demiranda Donna , Katula Karen S.
Published: September 2002

Overexpression of cyclin B has been detected in various human breast cancer cell lines, breast tumor tissues, and immortalized but nontransformed breast cells. The cause of this overexpression has not been thoroughly investigated, nor is it known if cyclin B protein forms a functional complex with its partner, cdk1, at inappropriate cell cycle periods. In this study we examined the pattern of cyclin B1 promoter activity in three breast cancer cell lines, BT‐549, MDA‐MB‐157, T‐47D, and the immortalized breast cell line MCF‐10F. Using cells stably transfected with a cyclin B1 promoter‐luciferase reporter, luciferase activity was measured throughout the cell cycle in lovastatin synchronized cells and in G1 and S/G2 phases of asynchronized cells by flow cytometry. Results demonstrate that the cyclin B1 promoter activity increases, as expected, during the S/G2 period in all the cell lines. However, some promoter activity can be detected in G1 phase of the different cell line with BT‐549 displaying the more altered pattern. Functional cyclin B1‐cdk 1 protein complex was detected in G1 phase of BT‐549 and T‐47D cell lines. These results suggest that in a subset of transformed breast cancer cells altered cyclin B1 promoter activity may contribute to the misexpression of cyclin B protein.
Genetic analysis of partial rust resistance in faba beans
Authors: Stoddard F. L. , Herath I. H. M. H. B.
Published: September 2002

Five parent populations of faba bean, differing in reaction to rust disease,
were crossed to prepare a complete diallel of F1
hybrids. Detached leaves of the parents and F1 hybrids
were challenged with 3 Australian rust populations and symptom development was
monitored from 11 to 16 days after inoculation, using a disease score related
to the percentage of leaf area covered with pustules, and a nominal value for
pustule size. Additive gene action was very important in determining the
response to rust disease, with ILB 3025 and ILB 3107 having similar low
disease scores, Fiord and Acc 327 having high values, and Icarus an
intermediate value. Cytoplasmic effects were also critical, with ILB 3025
having the most susceptible cytoplasm and ILB 3107 the most resistant, and
this was most readily detected in terms of the size of the pustule. Heterosis
conferred slightly lower disease scores and dominance was significant but of
lesser importance than additive gene action, particularly for disease score.
Rust population host gene action interactions were significant by the end of
the observation period, but were much smaller than the main effects. The
hybrid ILB 3107 ILB 3025 had less disease development than either parent,
indicating the potential for developing more resistant cultivars using
transgressive segregation. ILB 3107 shows particular promise for use as a
female line as it had both nuclear and cytoplasmic factors to contribute to
the development of resistant cultivars.
A regional adaptation analysis of oats in New South Wales and southern Queensland for grain yield and dry matter production
Authors: Herring M. R. , O'Brien L.
Published: September 2002

A regional adaptation analysis was conducted
to identify oat genotypes with potential for release as varieties or use as
parents in breeding programs for New South Wales (NSW) and southern
Queensland. This analysis used 5 trial sites (Toowoomba, Moree, Narrabri,
Armidale, Temora) and was conducted over 3 years using 71 oat
(Avena sp.) genotypes. Genotypes were selected to
represent various phenology types from early to late maturing as well as being
representative of a variety of end uses including grain, grazing, dual
purpose, and naked oats. Australian breeding lines and varieties composed most
of the genotypes in the analysis; however, overseas material was also
included.
Multivariate analysis indicated that the area studied could be divided into 2
subregions. The northern sites of Toowoomba, Moree, and Narrabri were in one
cluster, whereas Temora and Armidale formed another. Genotypes formed groups
which were largely related to their phenology. Results indicated that there
was a group of genotypes which produced above average grain yields over the
entire region. Oats in this group were primarily of mid-season phenology and
were all from the existing Australian oat breeding programs. Oats of mid- and
late-season phenology were better suited to the Armidale and Temora region
where conditions are cooler and the growing season longer. Early maturing oats
were identified as types suitable for high yielding grain oats for the
northern region. However, it is recognised that improvement in rust resistance
of these genotypes would be necessary for reliable and profitable grain
production in this area.
Breeding oats for northern NSW and southern Queensland would be possible using
genotypes from the existing Australian oat breeding programs. The quick
maturing oats from Agriculture Western Australia seemed especially well suited
to this purpose. From the genotypes used in this study there seemed to be a
shortage of late maturing oat types. If oats for hay or forage production were
required, evaluation of late maturing genetic material from overseas may be
required.