bluuebunny/crossref_metadata_2025 huggingface dataset:
A heat-shock-like response with cytoskeletal disruption occurs following hydrostatic pressure in MG-63 osteosarcoma cells
Authors: Haskin Christine L. , Athanasiou Kyriacos A. , Klebe Robert , Cameron Ivan L.
Published: December 2009

Human osteosarcoma cells, MG-63, were exposed to a hydrostatic pressure shock of 4.0 MPa for 20 min. Changes in subcellular distribution of the cytoskeletal elements and heat shock protein 70 (hsp70) were followed by indirect immunofluorescence and by avidin–biotin–peroxidase protocols. During recovery, total cellular RNA was determined and actin and aldolase mRNA content was followed using reverse transcription – polymerase chain reaction techniques. Hydrostatic pressure caused cell rounding (but not cell death), disruption of microtubules, collapse of intermediate filaments to a perinuclear location, collapse of actin stress fibers into globular aggregates in the cytoplasm, and the formation of several large elongated intranuclear actin inclusions. During recovery, the cells flattened, reorganized microtubules, and redistributed intermediate filaments prior to the reappearance of actin stress fibers. At 20 and 60 min following the initiation of hydrostatic pressure, there was increased anti-hsp70 staining at the nuclear membrane and concentration of hsp70 in four to six granules in the nucleus. At 120 min following the hydrostatic pressure, hsp70 showed intense staining in the cytoplasm and hsp70-containing granules in the nucleus disappeared. Cellular RNA decreased during the first 120-min posthydrostatic pressure shock and then recovered to near prehydrostatic pressure treatment levels by 240 min. Actin mRNA abundance, in relation to aldolase mRNA abundance, showed the same temporal pattern of initial decrease, followed by increase as did total RNA. Review of the literature indicated that eukaryotic cells respond to heat shock and to hydrostatic pressure by disruption of the cytoskeletal elements and by similar modifications in genetic expression. In this study, the observed responses of MG-63 cells to a 4-MPa hydrostatic pressure shock was like the reported response of mammalian cells to a 43 °C heat shock.Key words: heat shock response, hydrostatic pressure, cytoskeleton.
Effect of cholate on H+-ATPase and other proteins of dog renal brush-border membrane
Authors: Noël Josette , Fleser Angelica , Bellemare François , Thiéry Georges , Laprade Raynald , Burckhardt Gerhard , Vinay Patrick
Published: December 2009

A short treatment of dog renal brush-border membrane vesicles (BBMV) with sodium cholate, followed by dialysis of the detergent, reorients the polarity of H+-ATPase in the membrane and exposes its ATP binding sites to the extravesicular space, as previously shown with pig BBMV. In cholate-pretreated vesicles, the H+-ATPase remains fully active, but is inserted under the reversed polarity in sealed vesicles. A large spontaneous N-ethylmaleimide-sensitive ATPase activity is thus observed, as well as a steep intravesicular acidification upon external ATP addition, two findings absent in native vesicles. The ability of nitrate plus ATP to dissociate the hydrolytic subunits of the proton pump in cholate-pretreated vesicles, but not in native vesicles, demonstrates that most of the ATP binding subunits are accessible to ATP following cholate treatment. The sensitivity of the cytoplasmic domain of the H+-ATP activity to trypsin also confirms the reorientation of the enzyme in cholate-pretreated vesicles. The H+-ATPase and alkaline phosphatase remain largely associated with the membranes after the treatment with cholate, but γ-glutamyltranspeptidase, aminopeptidase N, and neutral endopeptidase are largely solubilized. Upon dialysis of cholate, all these enzymes are in part reinserted in the membrane according to their original polarity. The reorientation process is however specific for the H+-ATPase. Cholate treatment does not increase the formation of inside-out vesicles. Thus the treatment with cholate really reorients the polarity of the H+-ATPase in vesicles and allows for study of the proton pumping capacity of vacuolar H+-ATPase of proximal tubules.Key words: H+-ATPase polarity, kinetics, ectoATPases, brush-border membrane marker enzymes, vesicle polarity.
Stabilization in microsomal membranes of the fifth transmembrane segment of the Na+,K+-ATPase α subunit with proline to leucine mutation
Authors: Homareda Haruo , Kawakami Kiyoshi , Nagano Kei , Matsui Hideo
Published: December 2009

We have reported that the fifth transmembrane segment of the Na+,K+-ATPase α subunit could not be inserted into microsomal membranes when it was synthesized from the truncated α1 cDNA. In this study, three proline residues in the segment were changed into leucine residues by site-directed mutagenesis. Replacement of all three proline residues by leucine residues allowed the insertion of this segment and its translocation. This result confirms our previous finding and demonstrates that three proline residues in the fifth transmembrane segment prevent insertion of the segment into the membrane.Key words: Na+,K+-ATPase, transmembrane segment, membrane insertion, site-directed mutagenesis.
Assembly of adenovirus-specific nuclear inclusions in lytically infected HeLa cells: an ultrastructural and cytochemical study
Authors: Chaly Nathalie , Chen Xia
Published: December 2009

Adenoviruses (Ads) are nuclear DNA viruses that remodel host nuclear structure and function and induce formation of a variety of nuclear inclusions within which Ad DNA is replicated and transcribed. In this study, we have examined inclusion assembly by electron microscopy of samples stained conventionally or with bismuth to detect phosphoproteins. Small dense fibrillar bodies (DFBs) appeared very early associated with interchromatin granule (ICG) clusters. Somewhat later, similar DFBs lay near amorphous, loosely fibrillar structures that were moderately electron dense and showed little bismuth deposition. These clear fibrillar bodies (CFBs) enlarged and DFBs became embedded in their surface. At later stages, CFBs and DFBs were again dissociated. DFBs seen very early were poor in phosphoproteins, but later DFBs, whether embedded in the CFBs or lying near them, were intensely bismuth stained. DFBs and CFBs were less prominent once assembled virions were seen. At this late stage, virions were generally associated with moderately dense, slightly bismuth positive, irregularly shaped fibrillar inclusions that have previously been identified as viral genome storage sites. In addition, very dense fibrillar bodies, consisting usually of an electron-dense fibrillar shell and a less dense fibrogranular core, were observed at all but the earliest stages of infection, often at some distance from CFBs. There was also a major reorganization of host components during infection, including chromatin condensation, reduction of nucleolar volume and aggregation of the fibrillar regions at the nucleolar surface, and increased prominence of ICG clusters. A model is proposed for the assembly of Ad replication factories.Key words: adenovirus, lytic infection, replication factories, nuclear ultrastructure, cytochemistry.
Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes
Authors: Weaver Valerie M. , Lach Boleslaw , Walker P. Roy , Sikorska Marianna
Published: December 2009

Three chemically distinct serine, but not cysteine, protease inhibitors (phenylmethylsulphonyl fluoride, N-tosyl-L-phenylalanylchloromethyl ketone and 3,4-dichloroisocoumarin) prevented, in a dose-dependent manner, the characteristic apoptotic internucleosomal DNA cleavage (DNA ladder) typically observed in thymocytes in response to dexamethasone and teniposide VM-26. This effect was not the result of a direct inhibition of the Ca2+, Mg2+-dependent endonuclease, since oligonucleosomal DNA cleavage occurred in the presence of these inhibitors in isolated nuclei. The proteolytic step occurred at a very early stage of apoptosis, and preincubation of thymocytes with the inhibitors before dexamethasone or teniposide VM-26 were added irreversibly suppressed ladder formation. This implied that the cellular effector(s) of these compounds preexisted and were not resynthesized in response to the inducers of apoptosis. Serine protease inhibitors also suppressed apoptotic cell shrinkage and complete nuclear collapse, suggesting that these morphological changes were directly related to internucleosomal fragmentation of DNA. However, the serine protease inhibitors did not prevent high molecular weight DNA cleavage (> 50 kilobases) that preceded the ladder formation and thymocytes still died by apoptosis. This supported the view that internucleosomal DNA cleavage, considered to be the biochemical marker of apoptosis, might in fact be a late and dispensable step and that the newly described high molecular weight DNA cleavage might be a better indicator of apoptosis.Key words: serine protease, apoptosis, internucleosomal DNA fragmentation, high molecular weight DNA cleavage, protease inhibitors.
The effect of the heat shock response on ultrastructure of the centrosome of Drosophila cultured cells in interphase: possible relation with changes in the chemical state of calcium.
Authors: Marcaillou Christiane , Debec Alain , Lauverjat Sylvie , Saihi Armelle
Published: December 2009

Previous observations have shown that the heat shock response affects the centrosome function. We compared the ultrastructural organization of the centrosome in control (23 °C) and heat-shocked (37 °C, 50 min) interphase Drosophila cells to detect the nature of the lesions that could alter this organelle. The centrosome apparatus showed only minor modifications after the stress and the architecture of the centrioles appeared unaffected. The main difference concerned the organization of pericentriolar material which appeared more condensed and clotted. In extreme cases this material seemed to collapse on the centrioles. Recent reports proposed that Ca2+ concentrations could modify the distribution of pericentriolar material. In this study, we measured the changes in total and bound calcium in control or heat-shocked cell samples. The hyperthermia stress induced an increase of about 80% in global calcium. However, there was a decrease of about 50% in bound calcium. A heat shock stress seemed therefore to promote a change from the bound to the free state for a noticeable proportion of the element. As a preliminary hypothesis, these changes in the chemical state of calcium could be related to alterations in the pericentriolar material and thus with the functional inactivation of the centrosome. This view is also supported by calcium analysis on early Drosophila embryos. Contrary to cultured cells, Drosophila embryos did not present a stress inactivation of centrosomes. Equally, a heat shock did not disturb the bound calcium level in embryos.Key words: Centrosome, ultrastructure, calcium, heat shock, Drosophila.
A site-directed antibody recognizes a component of the ouabain-binding domain of the α1 subunit of rat Na+,K+-ATPase
Authors: Scully Robert R. , Pressley Thomas A. , O'Neil Roger G.
Published: December 2009

Antibodies (AbE1) were raised against an oligopeptide derived from a component of the ouabain-binding site of the α1 subunit of rat Na+,K+-ATPase. Preincubation of partially purified Na+,K+-ATPase from rat kidney with 1 μM AbE1 partially inhibited the enzyme and reduced its sensitivity to 1 mM ouabain. Antibodies against an unrelated protein were ineffectual. Unexpectedly, the masses of the proteins detected on immunoblots of rat kidney by AbE1, a 160 kilodalton (kDa) doublet, was greater than the 97 kDa of the α1 subunit. In lysates prepared with hot (85 °C) buffer constituted according to Laemmli's formulation for sample buffer (buffer L, a low chaotropic buffer), AbE1 always bound to the 160-kDa doublet. In contrast, AbE1 did not bind any proteins on blots of lysates that were prepared with buffer that contained greater concentrations of detergent and reducing agent (Laemmli's "homogenization" buffer, buffer H, a high chaotropic buffer), regardless of temperature. However, the presence of a, in lysates produced in homogenization buffer was confirmed by the presence of 97-kDa bands that were detected with two additional site-directed antibodies that recognize the α1 subunit. When homogenates prepared with sample buffer L were examined, one of these antibodies bound to bands of 160 kDa, indicating that α1 is a component of the bands bound by AbE1. It is concluded that AbE1 recognizes a component of the ouabain-binding site of Na+,K+-ATPase, but only under conditions in which the α1 subunit was associated into a higher molecular mass complex.Key words: isoenzyme, ouabain receptor, site-directed antibody.
Cell-swelling-induced taurine release from isolated perfused rat liver
Authors: Brand H. S. , Meijer A. J. , Gustafson L. A. , Jörning G. G. A. , Leegwater A. C. J. , Maas M. A. W. , Chamuleau R. A. F. M.
Published: December 2009

Astrocytes and lymphocytes are able to release significant amounts of taurine during periods of hypotonicity to reduce the increase in cell volume. To investigate this mechanism in the liver, we studied the release of free amino acids from isolated perfused rat liver during hypotonicity. The osmolarity of the perfusion medium was reduced from 305 to 255 or 205 mosM by decreasing the NaCl concentration 25 or 50 mM, respectively. This induced an 6–8% increase in liver mass and was associated with a specific 1.7-fold (−50 mosM) and 14-fold (−100 mosM) increase of the taurine release. None of the other amino acids measured showed a significant increase in their concentration in the effluent. The increase in taurine release occurred within 30 s after exposure to hypotonicity (maximal after 1–1.5 min) and followed closely the changes in liver mass. The taurine release declined gradually during successive exposures of the isolated liver to −100 mosM. This release was 29 and 17% of the original during the second and third exposure, respectively.Key words: cell swelling, liver, taurine.
Effect of hepatocyte swelling on microtubule stability and tubulin mRNA levels
Authors: Häussinger Dieter , Stoll Barbara , Dahl Stephan vom , Theodoropoulos Panayiotis A. , Markogiannakis Emmanuel , Gravanis Achille , Lang Florian , Stournaras Christos
Published: December 2009

Incubation of isolated rat hepatocytes under conditions known to induce cell swelling caused several alterations in microtubule physiology. As shown by immunofluorescence microscopy experiments in the absence and presence of triethyllead or colchicine (two well-established microtubule inhibitors), an apparent stabilization of the microtubule network became evident in hepatocytes exposed to hypotonic (190 mosmol/L) conditions. A similar stabilizing effect was also observed upon cell swelling induced by addition of insulin (100 nmol/L) or glutamine (10 mmol/L). The differential microtubule stabilities were not attributed to a differential incorporation of the antimicrotubular agents into hepatocytes as shown by [3H]colchicine-uptake experiments. The swelling-induced alterations of microtubules may contribute to the swelling-induced changes of liver cell function: in perfused rat liver it was found that the established inhibitory effect of hypotonic cell swelling on hepatic proteolysis was largely abolished in presence of colchicine. Tubulin mRNA levels increased by 1.9-, 2.1- and 2.7-fold in isolated hepatocytes being exposed for 120 min to hypotonic medium, insulin, or glutamine, respectively. The results suggest an involvement of microtubular structures in the regulation of liver metabolism in response to alterations of the cellular hydration state.Key words: microtubules, cell swelling, glutamine, gene expression, proteolysis.
Effect of tunicamycin and monensin on the transport to the cell surface and secretion of a viral membrane glycoprotein containing both N- and O-linked sugars
Authors: Ghosh-Choudhury Nandini , Butcher Martin , Reid Ed , Ghosh Hara P.
Published: December 2009

Most membrane glycoproteins contain either N-linked or O-linked oligosaccharides, which play important roles in correct folding, stability, and intracellular transport. Some glycoproteins, however, contain both the N- and O-linked sugars. To study the roles of the two types of glycosylation in intracellular transport we have used as a model the glycoprotein gC-1 of herpes simplex virus type 1 (HSV-1), which contains both N- and O-linked oligosaccharides. Cloned gene of gC-1 was expressed constitutively in mammalian cells to produce the gC-1 glycoprotein containing both types of glycosylation. Only a fraction of the gC-1 glycoprotein was secreted into the medium. Addition of tunicamycin blocked N-glycosylation and the gC-1 protein of reduced size containing only O-linked sugars was formed. This O-glycosylated gC-1 protein was transported to the cell surface and secreted into the medium, indicating that glycoprotein transport to and across the cell surface occurs in the absence of N-glycans. The data suggest either that O-glycans may contribute to this process or that transport can occur in the absence of both N- and O-glycans.Key words: intracellular transport, O-glycosylation, glycoprotein gC-1, herpes simplex virus type 1.
Decreasing expression of a gene encoding a protein related to basic fibroblast growth factor during differentiation of human preadipocytes
Authors: Teichert-Kuliszewska Krystyna , Hamilton Bradford S. , Deitel Mervyn , Roncari Daniel A. K.
Published: December 2009

Pursuant to our findings that pituitary basic fibroblast growth factor (bFGF) stimulates the replication of preadipocytes and inhibits their differentiation, we have studied the changes in expression of a bFGF-related mRNA during differentiation. Human omental preadipocytes were grown in primary culture and induced to differentiate with chemically defined serum-free medium. The differentiation process was assessed by monitoring the rise of glycerophosphate dehydrogenase (GPDH) activity, while mRNA expression for a bFGF-related protein(s) and GPDH was examined by amplifying the respective target sequences by polymerase chain reaction. In the case of all cell strains, differentiated preadipocytes revealed much lower expression of bFGF-related mRNA than undifferentiated preadipocytes. Concurrently, the expression of the GPDH mRNA rose significantly. The finding that the expression of the bFGF-related protein is decreased appreciably during adipose differentiation is consonant with its proposed function to expand and maintain adipose cells in a relatively undifferentiated state.Key words: preadipocytes, differentiation, basic fibroblast growth factor, obesity, glycerophosphate dehydrogenase.
Variations in serum levels of dolichol and its occurrence in human atherosclerotic plaques
Authors: Kothapalli Ravi , Guthrie Najla , Haines Milton D. S. , Carroll Kenneth K.
Published: December 2009

Nonsaponifiable lipids of human atherosclerotic plaques obtained at autopsy from patients ranging in age from 45 to 85 years were analyzed by high pressure liquid chromatography. All plaques contained dolichol, ranging from 125 to 460 μg/g wet weight. Dolichol was also present in normal aortic tissue, but the amounts were generally less than in plaques. To investigate the source of the dolichol in plaques, blood serum was analyzed from both volunteer subjects and hypercholesterolemic patients. The levels of dolichol were generally higher in hypercholesterolemic compared with normal subjects, but were not correlated with levels of total or lipoprotein cholesterol. The homologue pattern of dolichol in atherosclerotic plaques differed from that of aorta and blood. The source of dolichol in plaques and its significance remains to be established.Key words: dolichol, polyprenols, cholesterol, atherosclerosis, blood, plaques.
Recloning of SV40 early gene transfected human endothelial cells repeatedly recovers subpopulations with low passage characteristics and morphologies
Authors: Bailey Tina S. , Auersperg Nelly , Myrdal Sigrid E.
Published: December 2009

Cultured human umbilical vein endothelial cells (HUVECs) are a valuable model for investigation of endothelial functions, but they enter senescence at low passage. Transfection of early passage HUVECs with the early genes of SV40 greatly extends the replicative potential of these cells, but eventually results in marked changes in growth, morphology, and biochemistry. Here we report a modified approach that appears to have overcome the problem of late passage decline after transfection. Plasmid pX-8 containing the SV40 early genes was transfected into passage four HUVECs. At passage five, these transfectants were cloned by limiting dilution and selected on the basis of both morphological and biochemical resemblance to their untransfected counterparts. Two clones that expressed factor VIII and in which the basal and the tumor necrosis factor-α inducible levels of interleukin 6 and endothelial adhesion molecules were normal were chosen. Vimentin and fibronectin distribution in these clones resembled untransfected cells. At passage 25, growth pattern changes were becoming evident, but recloning these late passage clones recovered numerous subclones of normal, cobblestone appearance. Two of these were further characterized and found to resemble their original parental clone by all of the biochemical criteria listed above. These subclones appeared to transform more rapidly than the parental clone, but repeated subcloning again rescued clones with normal morphologies and normal biochemical characteristics. We conclude that periodic recloning may indefinitely perpetuate lines that are useful equivalents of their original counterparts.Key words: SV40, endothelial, immortalized, adhesion molecules.
Activities of acetylcholinesterase, choline acetyltransferase, and catecholamine production in the spinal cord of the axolotl Ambystoma mexicanum during forelimb regeneration
Authors: Scaps Patrick , Bernet François , Gautron Jean , Boilly Bénoni
Published: December 2009

Amputation of an axolotl limb causes severance of the brachial nerves, followed by their regeneration into a blastema. It is known that these nerves provide a neurotophic factor to blastemal cells. To approach the problem of the response of spinal cord nerve centers to forelimb amputation, we have studied biosynthetic activities in the nerve centers involved in axonal injury during limb regeneration. We report that the acetylcholinesterase (AChE) activity in the spinal cord is elevated 2 days (+ 69%) and 7 days (+ 28%) after limb amputation compared with levels in unamputated control animals, but is not significantly elevated at 3 h or 15 days. The percentages of slow (3.6 S and 6.0 S) and fast (18 S) sedimenting forms of AChE progressively decrease 2 and 7 days after amputation, while those of intermediate sedimenting forms (10.5 S and 14.0 S) increase. Fifteen days after amputation, lower molecular weight forms return to the control level, but the heavy molecular weight form of AChE is absent as at 7 days; consequently intermediate molecular weight forms are in a greater proportion than the other two forms. Choline acetyltransferase activity was measured only 2 days after amputation (when AChE was at its highest level). It increases by about 34% with regard to the controls. Adrenaline is higher than controls 2 days after amputation, while noradrenaline is not significantly modified. The metabolic changes observed in the spinal cord during limb regeneration probably are the result of a general reaction to the stress of amputation (transection of brachial nerves) and regeneration of nerve fibers, since similar metabolic activities were observed after a simple denervation of the two unamputated forelimbs.Key words: acetylcholinesterase, choline acetyltransferase, catecholamines, regeneration, axolotl.
Regulation of expression of the cyclic nucleotide phosphodiesterase gene in phosphodiesterase inhibitor-negative mutants of Dictyostelium discoideum
Authors: Adames Neil R. , Coukell M. Barrie , Wu Lin
Published: December 2009

During early development of Dictyostelium discoideum, the enzyme cyclic nucleotide phosphodiesterase (PD) is produced at a low rate during the period its specific inhibitor (PDI) is being synthesized. In addition, PD gene expression is derepressed in the aggregation-deficient (Agg−), Pdi− mutant HC35. These observations suggest that the PDI might function to regulate PD gene expression, as well as modulate its activity. To explore this idea further, five new Agg−, Pdi− mutants were isolated and analyzed. All of the mutants produced high PD activity and overexpressed PD mRNA; four exhibited elevated levels of the 2400-nucleotide aggregation transcript and one overproduced the 1900-nucleotide vegetative transcript. In contrast, PD transcripts were not elevated in two Agg−, Pdi+ mutants. To determine if PDI production regulates PD expression, HC35 cells were transformed with plasmids carrying the PDI structural gene under the control of either the vegetative or aggregative PD promoter. Neither expression of PDI by the transformants nor addition of partially purified PDI to HC35 cells affected PD transcription. These results suggest that PD overexpression in the Pdi− mutants is not a direct consequence of the inability of these cells to produce inhibitor.Key words: cAMP phosphodiesterase, phosphodiesterase inhibitor mutants, gene regulation, Dictyostelium.
Calmodulin: a versatile calcium mediator protein
Authors: Vogel Hans J.
Published: December 2009

The level of intracellular calcium is strictly regulated in all cells. In a resting cell, the [Ca2+] is ≤ 10−7 M and during activation it rises to approximately 10−6 M. Calmodulin (CaM) is the secondary messenger protein that has to translate this modest rise in intracellular calcium into a physiological response in all eukaryotic cells. CaM can activate almost 30 different target systems, including smooth muscle contraction, protein kinases and phosphatases, nitric oxide synthases, and calcium-extruding pumps. It is an acidic protein of 148 amino acids with four helix–loop–helix calcium-binding domains and it has a characteristic dumbbell shape in the crystal structure. In this review I discuss which features of CaM allow it to be such a universal and versatile calcium regulator. First of all, the positive cooperative calcium binding to all four binding sites of CaM in the presence of a target protein allows the protein to act effectively during a calcium transient. Secondly, the high Met content of two hydrophobic surface patches on the two domains of CaM creates a flexible and pliable, yet sticky, interaction surface that does not place high demands on the specificity of the interaction. Consequently, calcium-CaM can bind effectively to the CaM-binding domains of all its target proteins, despite their lack of amino acid sequence homology; their only common feature is that they are hydrophobic basic peptides that have a propensity to form an α-helix. CaM's capacity to recognize its CaM-binding domains is further enhanced by its third crucial feature, the intrinsic flexibility of the central linker region; this allows the two domains of CaM to slide over the surface of the α-helical bound peptide, to find their most favourable binding orientation. In this review I have also presented selected examples of a variety of experimental techniques that have contributed to our understanding of this unique multitasking protein. These include studies with well-established techniques such as site-directed mutagenesis, chemical modification, limited proteolysis, circular dichroism, and two-dimensional nuclear magnetic resonance (NMR), as well as novel or less common approaches involving the use of unnatural amino acids, metal-ion NMR, lysine pKa determinations, and isotope-edited Fourier transform infrared spectroscopy. In combination with available structural information, these studies have provided considerable detail in our understanding of this versatile calcium regulatory protein.Key words: calmodulin, calcium metabolism, methionine, lysine, NMR spectroscopy.
cAMP regulation of phenobarbital-mediated induction of δ-aminolevulinate synthase mRNA in hepatocytes from normal and experimental diabetic rats
Authors: Varone Cecilia L. , Canépa Eduardo T. , Llambías Elena B. C. , Grinstein Moisés
Published: December 2009

We examined the mechanism underlying the effect of cAMP on δ-aminolevulinate synthase mRNA biosynthesis in isolated hepatocytes from normal and experimental diabetic rats. We have demonstrated that the potentiation by dibutyryl cAMP of the phenobarbital-mediated induction of δ-aminolevulinate synthase enzyme activity, observed in our previously reported studies, reflects an increased amount of its mRNA. The inducing effect exerted by phenobarbital on the biosynthesis of δ-aminolevulinate synthase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response to the increased level of endogenous cAMP in diabetic hepatocytes is apparently sufficient for a maximum activation of the cAMP-dependent protein kinase. The present results suggest that in rat liver dibutyryl cAMP modulates δ-aminolevulinate synthase mRNA biosynthesis by acting predominantly, if not exclusively, at the level of gene transcription.Key words: δ-aminolevulinate synthase mRNA, phenobarbital, cAMP, diabetic rat hepatocytes.
Influence of exogenous growth factors on the synthesis and secretion of collagen types I and III by explants of normal and healing rabbit ligaments
Authors: Murphy Patricia G. , Loitz Barbara J. , Frank Cyril B. , Hart David A.
Published: December 2009

In this investigation it has been demonstrated that specific growth factors are able to modify collagen secretion in explants from healing rabbit medial collateral ligaments. The addition of 2.5 ng transforming growth factor β1 (TGF-β1)/mL to 3-week-old scar explants resulted in an increase in the total amount of collagen secreted. Analysis of collagen types I and III individually revealed that the increase mediated by TGF-β1 was due primarily to an increase in collagen type I secretion. This led to a ratio of type I : type III that is closer to that found in normal ligament tissue. The addition of 100 ng insulin-like growth factor 2 (IGF-2) to explant cultures of 3-week-old scar tissue also led to an increase in the quantity of collagen secreted, but the increase was in both type I and III collagens. These effects were observed to a lesser degree in 6-week-old scar tissue, and by 12 weeks postinjury, minimal effects of the growth factors on collagen synthesis was detected. Neither growth factor influenced collagen secretion by normal ligament or synovium. In contrast, IGF-1 (100 ng/mL) or basic fibroblast growth factor (bFGF) (10 ng/mL) did not exert a detectable effect on collagen secretion by any of the normal or healing tissues. These results indicate that TGF-β1 and IGF-2 can modify the metabolic activity of cells in explants of healing ligaments early after injury and may enhance the repair process leading to improved function.Key words: ligament healing, transforming growth factor β1, insulin-like growth factors 1 and 2, basic fibroblast growth factor, collagen synthesis.
Expression of candidate cell death genes in cell lines during apoptosis
Authors: Häcker Georg , Vaux David L.
Published: December 2009

We have cloned mouse candidate cell death genes RP-2 and nedd2 and used Northern blot analysis to study their expression in a growth-factor-dependent cell line (FDC-P1) that can be induced to undergo apoptosis by growth factor withdrawal and in a thymoma line (ST4) that undergoes apoptosis when irradiated. RP-2 was expressed in FDC-P1 cells even when not undergoing apoptosis, and mRNA levels did not increase when apoptosis was induced by growth factor withdrawal. FDC-P1 cells expressed two genes that are similar to the Caenorhabditis elegans cell death gene ced-3: a 3.7 kilobase (kb) nedd2 message and low levels of a 5-kb message for interleukin-1β converting enzyme. Levels of these messages did not change with the induction of cell death, but transfection of fibroblasts with constructs expressing nedd2 caused them to undergo apoptosis. These results suggest that expression of RP-2 and nedd2 is not sufficient for apoptosis, and if the products of these genes are involved in apoptosis of FDC-P1 cells, they are regulated by posttranslational mechanisms.Key words: apoptosis, RP-2, nedd2, interleukin-1β converting enzyme.
Increased levels of clusterin mRNA in the ventral prostate of the aging rat are associated to increases in cuboidal (atrophic) cell population and not to changes in apoptotic activity
Authors: Marinelli Maria , Quaglino Daniela , Bettuzzi Saverio , Strocchi Paola , Davalli Pierpaola , Corti Arnaldo
Published: December 2009

In the ventral prostate of the intact rat, clusterin mRNA is expressed in a small population of cuboidal epithelial cells undergoing apoptosis, but not in the columnar cells comprising the vast majority of the glandular epithelium. Upon castration, clusterin mRNA expression and apoptotic activity are turned off in the cuboidal cells and turned on in the columnar ones. We show here that the progressive enhancements in the abundance of clusterin mRNA, occurring in the rat ventral prostate upon aging, are based on increases of the cuboidal cells at the expense of the columnar ones. DNA fragmentation, a typical sign of apoptosis, assayed both by agarose gel electrophoresis and in situ staining, was undetectable in 3-month-old rats but was evident among the cuboidal cells of 24-month-old animals and columnar cells of 1-day castrates. The DNA content of the ventral prostate did not change significatively between young and old rats, indicating that no increase in the rate of cell death occurs within the age interval examined. It is concluded that the enhancement in cuboidal cell population, the consequent augmented accumulation of clusterin mRNA, and the increased frequency of DNA fragmentation that we have detected in the aging rat ventral prostate are not directly related to the rate of apoptosis.Key words: apoptosis, clusterin, rat ventral prostate.
The role of growth factors in the suppression of active cell death in the prostate: an hypothesis
Authors: Guenette R. Sean , Tenniswood Martin
Published: December 2009

Regression of the rat ventral prostate occurs when the level of 5α-dihydrotestosterone, the trophic hormone, drops below the threshold required to suppress apoptosis. The induction of apoptosis in the ventral prostate is accompanied by the increase in the steady-state level of a number of mRNAs coding for proteins that are involved in the latter stages of apoptosis and thus represent secondary thanatogens. These include proteases (cathepsins, plasminogen activators, and collagenase), clusterin, poly(ADP)ribose polymerase, tenascin, and several unidentified genes, as well as several RNases and the classical Ca2+,Mg2+-dependent endonuclease. In addition, insulin-like growth-factor-binding protein 5 (IGFBP-5) is induced de novo. We propose that IGFBP-5 may serve to trigger the apoptotic process through the attenuation of the insulin-like growth factor signalling system (which is necessary for cell survival), and as such, represents a primary thanatogen in the prostate.Key words: insulin-like growth factor, insulin-like growth-factor-binding proteins, prostate, apoptosis, regression, gene expression.
Programmed cell death: early changes in metamorphosing cells
Authors: Lockshin Richard A. , Zakeri Zahra
Published: December 2009

Programmed cell death in intersegmental muscles and labial glands of Manduca sexta is a type II form of active cell death, in which lysosomal destruction of cytoplasm is a prominent and early feature, and the collapse of the nucleus is late and relatively modest until the bulk of the cytoplasm has been eroded. The prominent features of this collapse include an early expansion of the lysosomal compartment and a decrease in overall protein synthesis, while a small number of mRNAs persist or are upregulated. Energy resources appear to be adequate during the early stages of degeneration, and changes in levels of second messages likewise do not seem to be sufficiently dramatic to explain the failure of the tissue. There is some suggestion that DNA may not be completely intact long before the labial gland finally collapses, suggesting that it may fail under heavy load. Otherwise, we do not yet have an explanation for the precipitous drop in overall protein synthesis.Key words: programmed cell death, apoptosis, lysosomes, endonuclease, protein synthesis, Manduca sexta.
DNA cleavage in rat liver nuclei activated by Mg2+ or Ca2+ + Mg2+ is inhibited by a variety of structurally unrelated inhibitors
Authors: Cain Kelvin , Inayat-Hussain Salmaan H. , Kokileva Ludmilla , Cohen Gerald M.
Published: December 2009

Internucleosomal DNA fragmentation is often regarded as the biochemical hallmark of apoptosis and can be reproduced in vitro in rat liver nuclei. In this study we demonstrate that DNA is initially cleaved into ≥700, 200–250, and 30–50 kilobase pair (kbp) fragments via a Mg2+-dependent, multistep process which can be potentiated by Ca2+. The subsequent internucleosomal cleavage requires both Ca2+ and Mg2+. Furthermore, we show that the heavy metals Cd2+ and Hg2+, dichloroisocoumarin (a general serine protease inhibitor), and N-ethyl maleimide (NEM, a specific thiol reagent) are potent inhibitors of both the Mg2+- and Ca2+/Mg2+-stimulated DNA fragmentation. In contrast, two other serine protease inhibitors, N-α-tosyl-L-lysine chloromethylketone and N-tosyl-L-phenylalanine chloromethylketone are weak and ineffective, respectively, as inhibitors of DNA cleavage. Increasing inhibition of DNA cleavage is accompanied by a shift in the size of the cleaved DNA fragments, which increases from mono- + oligo-nucleosomes → 30–50 kbp → 200–300 kbp → ≥ 700 kbp → intact DNA. Dithiothreitol, a dithiol, blocks NEM and dichloroisocoumarin inhibition, and since Cd2+ and Hg2+ are also potent —SH blocking agents it is proposed that a critical thiol is involved in the cleavage of DNA into both large kbp fragments and oligonucleosomal-sized fragments.Key words: DNA cleavage, protease inhibitors, thiols.
Degradation of constitutive transcription factors during apoptosis in rat thymocytes
Authors: Belle Ian de , Testolin Lucia , Pandey Siyaram , Carson Christine , Walker P. Roy , Armato Ubaldo , Sikorska Marianna
Published: December 2009

Dexamethasone (Dex) accelerates the rate of apoptosis in thymocytes by a process thought to require gene expression. Among the genes implicated in the regulation of this phenomenon are the immediate early genes such as c-fos and c-jun, whose expression is modulated by a complement of preexisting transcription factors. We have analyzed the DNA-binding activity of these constitutive transcription factors during Dex-induced apoptosis in thymocytes to assess their functionality. We observed a progressive loss of the DNA-binding proteins in parallel with the appearance of the characteristic morphological and biochemical features of apoptosis. At the same time we have found a general increase in the nuclear proteolytic activity concomitant with a significant loss of the nuclear nonhistone chromosomal proteins. Indeed, cotreatment of thymocytes with the nonspecific serine protease inhibitor phenyl-methylsulphonyl fluoride was able to partially protect the stability of the DNA-binding proteins and alter the expression of the c-fos and c-jun genes but did not inhibit apoptosis. Our results suggest that the action of a protease(s) is responsible for the degradation of constitutive transcription factors during Dex-induced apoptosis, rendering the death pathway irreversible.Key words: apoptosis, thymocytes, proteolysis, transcription factors, gene expression.
The use of C0t-1 probe DNA for the detection of low levels of DNA fragmentation
Authors: Wong Paul , Smith Sylvia B. , Bora Naba , Gentleman Susan
Published: December 2009

In many apoptotic systems the final demise of the DNA results in the generation of multinucleosomal-sized DNA fragments, which appear as a DNA ladder after agarose electrophoresis. Extensive DNA fragmentation can be detected by ethidium bromide staining. Visualization of low levels of DNA fragmentation, however, requires both a more sensitive detection system, as well as a method of DNA extraction, that limits the extent of high molecular weight DNA shearing. We have found that the use of C0t-1 repetitive DNA as a probe for Southern analysis of DNA ladders is a sensitive method to detect low levels of DNA fragmentation. We have applied this methodology in the detection of DNA fragmentation in the normal developing C57BL/6 mouse retina at stages in which there is known DNA fragmentation, as well as in human Y-79 retinoblastoma cells grown in culture. We have found that in many instances in which there is no detectable DNA ladder with ethidium bromide staining a very definitive DNA ladder can be visualized via Southern blot analysis with a C0t-1 DNA probe.Key words: DNA fragmentation, apoptosis, C0t-1.
The binding of analogues of coralyne and related heterocyclics to DNA triplexes
Authors: Latimer Laura J. P. , Payton Natasha , Forsyth Gavin , Lee Jeremy S.
Published: December 2009

Coralyne has been shown previously to bind well to both T∙A∙T- and C∙G∙C+-containing triplexes. Derivatives of coralyne were prepared and their binding to poly(dT)∙poly(dA)∙poly(dT) and poly[d(TC)]∙poly[d(GA)]∙poly[d(C+T)] was assessed from thermal denaturation profiles. A tetraethoxy derivative showed only weak binding to both types of triplex. Analogues with extended 8-alkyl chains showed good binding to poly(dT)∙poly(dA)∙poly(dT), but the preference for triplex poly[d(TC)]∙poly[d(GA)]∙poly[d(C+T)] was decreased compared with the duplex. Sanguinarine, a related alkaloid, bound well to poly(dT)∙poly(dA)∙poly(dT) but only weakly to the protonated triplex. It is hypothesized that the position of the protonated nitrogen ring is important for binding to poly[d(TC)]∙poly[d(GA)]∙poly[d(C+T)]. A series of other chromophores was studied and only those with a positive charge bound to triplexes. All of these bound well to poly(dT)∙poly(dA)∙poly(dT) but only weakly if at all to the duplex poly(dA)∙poly(dT). In contrast, most of them did not bind well to the triplex poly[d(TC)]∙poly[d(GA)]∙poly[d(C+T)] and those that did still showed a preference for duplex poly[d(TC)]∙poly[d(GA)]. In general, preference for triplex poly(dT)∙poly(dA)∙poly(dT) compared with the duplex is a common feature of intercalating drugs. On the other hand, specificity for protonated triplexes may be very difficult to achieve.Key words: triplex DNA, DNA-binding drugs, intercalation.
Purification and characterization of a new α-amylase of intermediate thermal stability from the yeastLipomyces kononenkoae
Authors: Prieto José Antonio , Bort Bernardo Roque , Martínez Javier , Randez-Gil Francisca , Sanz Pascual , Buesa Carlos
Published: December 2009

A new α-amylase from the extracellular culture of the yeast Lipomyces kononenkoae CBS 5608 has been purified to homogeneity by ammonium sulphate treatment, affinity binding on cross-linked starch, and DEAE–Biogel A chromatography. The enzyme was monomeric, with an apparent Mrof 76 kilodaltons, pI < 3.5, and optimum pH 4.5–5.0, and exhibited intermediate thermal stability. The temperature for optimal enzyme activity was 70 °C. It is a glycoprotein with both N- and O-linked sugars. Kinetic analyses indicate that the enzyme has an endoamylolytic mechanism. The kMfor soluble starch was 0.80 g∙L−1and the kcatwas 622∙s−1. Keywords: α-amylase, glycosylation, intermediate thermal stability, Lipomyces kononenkoae.
ADictyostelium discoideumgene, which is highly related tomo15fromXenopus, is expressed during growth but not during development
Authors: Michaelis Christine , Luo Qian , Weeks Gerald
Published: December 2009

We have isolated a cDNA from the cellular slime mold Dictyostelium discoideum encoding a protein that is 52% identical to the Xenopus Mo15 kinase and highly related to the equivalent proteins from human (52% identity), rice (52.7% identity), and yeast (47.6% identity). Mo15 is responsible for the activation of Cdc2 kinase and is itself a member of the large Cdc2-related family of protein kinases. The Dictyostelium protein is more related to the Xenopus Mo15 protein than it is to either the Dictyostelium Cdc2 or Crp proteins. Southern blot analysis of genomic V12-M2 DNA indicated that mo15 is present as a single copy gene that cross hybridizes with cdc2 at low stringency. Northern blot analysis of RNA from different stages of Dictyostelium development showed that mo15 is only expressed during vegetative cell growth.Key words: cell cycle, differentiation, cell type determination, Cdc2 kinase, Dictyostelium, Mo15 protein.
Inhibition of hydroxymethylglutaryl coenzyme A reductase activity does not affect the secretion rate of apolipoproteins B and AI by CaCo-2 cells
Authors: Pau Ellick , He Yi , Lougheed Marilee , Steinbrecher Urs P.
Published: December 2009

It is believed that the major mechanisms by which hydroxymethylglutaryl coenzyme A reductase inhibitors lower plasma cholesterol levels are by inducing hepatic low-density lipoprotein (LDL) receptor activity and by decreasing apolipoprotein B (apoB) secretion by the liver. However, the intestine is also an important cholesterogenic organ and the possibility that this class of drugs may alter lipoprotein secretion by the intestine has not been fully studied. The purpose of the present study was to examine the possible role of cholesterol in regulating apoB secretion by the intestine by testing if the suppression of cholesterol synthesis by the reductase inhibitor lovastatin affected the secretion of apoB by CaCo-2 human intestinal cells. Differentiated post-confluent CaCo-2 cells were incubated for 24–72 h in serum-free medium in the presence or absence of 5 μM lovastatin, and the secretion rate of lipids, as well as apoB and apolipoprotein AI (apo AI) into the medium, was measured. Lovastatin markedly inhibited the incorporation of [1-14C]acetate into cholesterol for at least 48 h, lowered the content of esterified cholesterol in cells, and reduced their rate of cholesterol secretion. However, under basal conditions lovastatin had no effect on the secretion rate of apoB. After stimulation of apoB secretion by addition of 0.8 mM oleic acid to the medium, lovastatin did not alter apoB secretion in the first 2 days of incubation, but reduced the content of apoB in media from the 3rd day by 30%. This could not be explained by an increase in the rate of LDL degradation. Furthermore, supplementation with mevalonic acid only reversed about one-half of the effect of lovastatin, suggesting that this effect was at least partly nonspecific or unrelated to inhibition of cholesterol biosynthesis. There was also no specific effect of lovastatin on apoAI secretion. When cells were cultured with [1-14C]acetate for 24 or 72 h, the specific activity of cholesterol in medium at the end of the incubation was the same as in cells, suggesting that cholesterol used for lipoprotein secretion was in equilibrium with bulk cellular cholesterol and was not from a segregated compartment derived from newly synthesized cholesterol. This may explain why apoB secretion by CaCo-2 cells was unaffected by inhibition of cholesterol synthesis with lovastatin.