Particulate Subcellular Fractions Research Articles

Partitioning of paralytic shellfish toxins in sub-cellular fractions of the digestive gland of the cockle Cerastoderma edule: Changes under post-bloom natural conditions

Concentrations of paralytic shellfish toxins (C1+2, B1, dcGTX2+3, dcSTX, GTX2+3 and STX) were determined by LC-FLD in composite samples of digestive glands of the cockle Cerastoderma edule and in each sub-cellular particulate fractions obtained after differential centrifugation (nuclei+debris, mitochondria, lysosomes and microsomes). The specimens were sampled during the exposure to a bloom of Gymnodinium catenatum (day 0) and in the subsequent 8, 12, 14, 19, 21 and 25 days under natural depuration conditions. Toxin profiles of digestive glands were dominated by C1+2 followed by B1 and dcGTX2+3, although the proportion between C1+2 and B1 contents decreased with the time, indicating a slower elimination of B1. All toxins, except GTX2+3 and STX, were quantified in the four sub-cellular fractions. The content of the quantified toxins decreased most markedly in nuclei+debris and microsomal fractions, during the first eight and 12 days, respectively. Conversely, different patterns were observed among toxins in mitochondrial and lysosomal fractions. The less accentuated decreases of dcGTX2+3 and dcSTX contents in the mitochondrial fraction may have resulted from the conversion of other toxins, like C1+2 and B1, associated with enzymatic activities in that fraction. The largest discrepancy was registered in lysosomal fraction for B1, since its content increased after eight days of post-bloom conditions. Input of B1 may come from the conversion of other toxins, like the abundant B2 and C1+2. These transformations are associated to the major role of lysosomes in the intra-cellular digestive process of materials acquired through vesicular transport.

Biodynamics, Subcellular Partitioning, and Ultrastructural Effects of Organic Selenium in a Freshwater Bivalve

Selenium is a trace element characterized by concentrations that narrowly range between being essential and being toxic. Even though inorganic selenite and selenate are the predominantchemicalforms ofSe in surfacewaters, the toxicity of Se to aquatic organisms is mostly governed by the bioavailability of organic selenium within food webs. The present study was designed to evaluate organic selenium bioaccumulation and toxicity patterns in the freshwater sentinel species Corbicula fluminea. Waterborne selenomethionine (SeMet) exposure was used to mimic dietary organo-Se uptake. Our results demonstrate that SeMet is accumulated to a relatively high extentwith a concentration factor of 770 (wet weight basis). Higher uptake than depuration rates suggest that bivalves deal with high Se amounts using a strategy of detoxification based on Se sequestration that could involve granules, as shown by a strong increase of Se in the particulate subcellular fraction. Selenium is persistent in the cytosol of bivalves exposed to SeMet where it is found in proteins of a wide range of molecular mass, indicating a possible replacement of methionine by selenomethionine. A subsequent alteration of protein function might be one of the mechanisms of Se toxicity that could explain the histopathological effects we observed in gills by using transmission electronic microscopy. Those analyses showed changes in gill filament ultrastructure and suggested mitochondria asthefirsttargetfor SeMet cytotoxicity, with alterations of the outer membrane and of cristae morphology. Organo-Se would thus not only be toxic via indirect mechanisms of maternal transfer as it was suggested for fish but also directly. Our results on Se distribution agree with studies that used dietary Se transfer, and highlight the relevance (and less expensive way) of using SeMet water-only exposure protocols to mimic the real environment.

Expression of the cAMP-Phosphodiesterase PDE4D Isoforms and Age-Related Changes in Follicle-Stimulating Hormone-Stimulated PDE4 Activities in Immature Rat Sertoli Cells1

Major changes in the cAMP-dependent signal transduction pathway triggered by FSH take place during transition of rat Sertoli cells from proliferative to the quiescent/terminally differentiated state. Using Sertoli cell cultures isolated from 10-, 20-, and 30-day-old rats, we recorded a specific increase in PDE4 activity in both the soluble and particulate subcellular fractions of 20-day-old Sertoli cells, which also displayed the highest cAMP response to FSH and the highest FSH-induced increase in PDE4 activity in both subcellular compartments. RT-PCR and immunoblotting experiments showed that almost all the PDE4D isoforms, known as the main cAMP-regulated rolipram-sensitive PDE in Sertoli cells, were expressed throughout the early postpartum period, whereas only the short PDE4D isoforms (PDE4D1 and PDE4D2) were transcriptionally regulated by FSH. Unexpectedly, the immunoblot data also revealed that the soluble PDE4 activities were mainly related to the long PDE4D isoforms and that short PDE4D1 was predominantly particulate. The subcellular distribution and expression of PDE4D proteins were unaffected by the developmental status of the Sertoli cells. Only the expression of short PDE4D1 appeared to be upregulated by FSH and only in 20-day-old Sertoli cells, which suggests phenotype-dependent differential regulation of Pde4d1 mRNA translation. Resensitization of the cAMP response to FSH in 20-day-old Sertoli cells was also associated with the highest FSH-induced transient increase in both soluble and particulate PDE4 activities, which suggests developmental changes in the PKA-mediated upregulation of the catalytic activities of long PDE4D. Such alterations may be involved in the phenotype-dependent alterations in FSH receptor coupling with its associated G proteins in rat Sertoli cells.

Role of glycogen content in insulin resistance in human muscle cells

We have used primary human muscle cell cultures to investigate the role of glycogen loading in cellular insulin resistance. Insulin pre-treatment for 2 h markedly impaired insulin signaling, as assessed by protein kinase B (PKB) phosphorylation. In contrast, insulin-dependent glycogen synthesis, glycogen synthase (GS) activation, and GS sites 3 de-phosphorylation were impaired only after 5 h of insulin pre-treatment, whereas 2-deoxyglucose transport was only decreased after 18 h pre-treatment. Insulin-resistant glycogen synthesis was associated closely with maximal glycogen loading. Both glucose limitation and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) treatment during insulin pre-treatment curtailed glycogen accumulation, and concomitantly restored insulin-sensitive glycogen synthesis and GS activation, although GS de-phosphorylation and PKB phosphorylation remained impaired. Conversely, glycogen super-compensation diminished insulin-sensitive glycogen synthesis and GS activity. Insulin acutely promoted GS translocation to particulate subcellular fractions; this was abolished by insulin pre-treatment, as was GS dephosphorylation therein. Limiting glycogen accumulation during insulin pre-treatment re-instated GS dephosphorylation in particulate fractions, whereas glycogen super-compensation prevented insulin-stimulated GS translocation and dephosphorylation. Our data suggest that diminished insulin signaling alone is insufficient to impair glucose disposal, and indicate a role for glycogen accumulation in inducing insulin resistance in human muscle cells.

Naturally Occurring DNA Transfer System Associated with Membrane Vesicles in Cellulolytic Ruminococcus spp. of Ruminal Origin

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.

TRICHOMONAS VAGINALIS: IDENTIFICATION OF SOLUBLE AND MEMBRANE-ASSOCIATED PHOSPHOLIPASE A1AND A2ACTIVITIES WITH DIRECT AND INDIRECT HEMOLYTIC EFFECTS

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthal's inhibitor. The greatest effect was observed with 80 microM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.

Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel.

The effects of short-term burst (5 min at 1.8 m/s) swimming and long-term cruiser (60 min at 1.2 m/s) swimming on maximal enzyme activities and enzyme distribution between free and bound states were assessed for nine glycolytic and associated enzymes in tissues of horse mackerel, Trachurus mediterraneus ponticus. The effects of exercise were greatest in white muscle. The activities of phosphofructokinase (PFK), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase), and phosphoglucomutase (PGM) all decreased to 47, 37, 37 and 67%, respectively, during 60-min exercise and all enzymes except phosphoglucoisomerase (PGI) and PGM showed a change in the extent of binding to subcellular particulate fractions during exercise. In red muscle, exercise affected the activities of PGI, FBPase, PFK, and lactate dehydrogenase (LDH) and altered percent binding of only PK and LDH. In liver, exercise increased the PK activity 2.3-fold and reduced PGI 1.7-fold only after 5 min of exercise but altered the percent binding of seven enzymes. Fewer effects were seen in brain, with changes in the activities of aldolase and PGM and in percent binding of hexokinase, PFK and PK. Changes in enzyme activities and in binding interactions with subcellular particulate matter appear to support the altered demands of tissue energy metabolism during exercise.

Metabolic fate of exogenous diacylglycerols in A10 smooth muscle cells

The metabolic fate of exogenous diacylglycerols, 1-palmitoyl-2- [1- 14 C] oleoyl- sn-glycerol ( 2-[ 14 C] POG) and 1-stearoyl-2- [1- 14 C] arachidonoyl- sn-glycerol (2- [ 14 C] SAG), was determined after incubation of A10 smooth muscle cells with liposomal suspensions. Hydrolysis through a diacylglycerol (DG) lipase pathway was the predominant metabolic fate; more than 80% of cell-associated radioactivity from 2-[ 14 C] POG and 2-[ 14 C] SAG was recovered in lipolytic products, monoacylglycerol (MG) and fatty acids (FA), which were present in the incubation medium. Hydrolysis of 2-[ 14 C] POG was reduced completely by tetrahydrolipstatin, a lipase inhibitor. Very little radioactivity from either 2-[ 14 C] POG or 2-[ 14 C] SAG was incorporated into triacylglycerol or phospholipids. DG lipase and kinase activities were measured by in vitro enzyme assays. 1-[1- 14 C] Palmitoyl-2-oleoyl- sn-glycerol ( 1-[ 14 C] POG) was phosphorylated (kinase activity) to a greater extent than 2-[ 14 C] SAG in assays with both soluble and particulate subcellular fractions from A10 cells. DG lipase activity (hydrolysis of 1-[ 14 C] POG and 2-[ 14 C] SAG) was markedly stimulated by the addition of 20 mM MgCl 2 and 20 mM ATP to the assay. Under optimal assay conditions, DG lipase activity exhibited little substrate specificity. Our findings indicate that exogenous DG are mainly hydrolyzed by DG and MG lipases in A10 smooth muscle cells; as a result, signalling mechanisms responding to DG second messengers will be attenuated.

Cardiorespiratory responses to systemic administration of a protein kinase C inhibitor in conscious rats.

Although protein kinase C (PKC) is an essential component of multiple neurally mediated events, its role in respiratory control remains undefined. The ventilatory effects of a systemically active PKC inhibitor (Ro-32-0432; 100 mg/kg i.p.) were assessed by whole body plethysmography during normoxia, hypoxia (10% O2), and hyperoxia (100% O2) in unrestrained Sprague-Dawley rats. A sustained expiratory time increase occurred within 8-10 min of injection in room air[mean 44.8 +/- 5.2 (SE) % ], was similar to expiratory time prolongations after Ro-32-0432 administration during 100% O2 (45.5 +/- 8.1%; not significant), and was associated with mild minute ventilation (VE) decreases. Hypercapnic ventilatory responses (5% CO2) remained unchanged after Ro-32-0432. During 10% O2, VE increased from 122.6 +/- 15.6 to 195.7 +/- 10.1 ml/min in vehicle-treated rats (P < 0.001). In contrast, marked attenuation of VE hypoxic responses occurred after Ro-32-0432 [86.2 +/- 6.2 ml/min in room air to 104.1 +/- 7.1 ml/min in 10% O2; pre- vs. post-Ro32-0432, P < 0.001 (analysis of variance)]. Overall, PKC activity was reduced and increases with hypoxia were abolished in the particulate subcellular fraction of brain tissue after Ro-32-0432 treatment, indicating that this compound readily crosses the blood-brain barrier. We conclude that systemic PKC inhibition elicits significant centrally mediated expiratory prolongations and ventilatory reductions as well as blunted ventilatory responses to hypoxia but not to hypercapnia. We postulate that PKC plays an important role in signal transduction pathways within brain regions underlying respiratory control.

Characterization of Phosphotyrosine Phosphatase Activity in Sheep Platelets: Amphiphilic and Hydrophilic Forms

Using O-phosphotyrosine as a substrate, we characterized the phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity from sheep platelets. PTPase was found to be located in three particulate subcellular fractions and in the cytosol, with K m values in the millimolar range. PTPase was strongly inhibited by vanadate, molybdate and HgCl 2 and only weakly inhibited by Zn 2+. Other divalent cations and NaF had no significant effect on the activity associated with the membrane fraction but were slightly stimulatory as regards cytosolic activity. Heparin inhibited cytosolic activity 2-fold more than membrane-bound activity and dithiothreitol only inhibited cytosolic PTPase. Polycationic compounds were seen to be weak stimulators of all the PTPase activity. Solubilization of the PTPase from membranes always required a detergent. When subjected to Triton X-114 phase partitioning, PTPase was recovered in the detergent-rich (35%) and in the detergent-poor (65%) phases. Sedimentation analysis of the cytosolic PTPase showed a peak of 3.2S that remained unmodified when Triton X-100 or Brij 97 sucrose gradients were used. Sedimentation analysis of the membrane-associated PTPase showed 6S and 3.7S peaks unchanged in Triton X-100 or Brij 97 gradients together with 7.5S and 10.3S shoulders that shifted to smaller sedimentation coefficients in Brij 97 sucrose gradients. These results support the view that sheep platelets contain amphiphilic and hydrophilic forms of PTPase.

Protein Kinase C Isozyme-mediated Cell Cycle Arrest Involves Induction of p21waf1/cip1 and p27kip1 and Hypophosphorylation of the Retinoblastoma Protein in Intestinal Epithelial Cells

The molecular mechanisms underlying protein kinase C (PKC) isozyme-mediated control of cell growth and cell cycle progression are poorly understood. Our previous analysis of PKC isozyme regulation in the intestinal epithelium in situ revealed that multiple members of the PKC family undergo changes in expression and subcellular distribution precisely as the cells cease proliferating in the mid-crypt region, suggesting that activation of one or more of these molecules is involved in negative regulation of cell growth in this system (Saxon, M. L., Zhao, X., and Black, J. D. (1994) J. Cell Biol. 126, 747-763). In the present study, the role of PKC isozyme(s) in control of intestinal epithelial cell growth and cell cycle progression was examined directly using the IEC-18 immature crypt cell line as a model system. Treatment of IEC-18 cells with PKC agonists resulted in translocation of PKC alpha, delta, and epsilon from the soluble to the particulate subcellular fraction, cell cycle arrest in G1 phase, and delayed transit through S and/or G2/M phases. PKC-mediated cell cycle arrest in G1 was accompanied by accumulation of the hypophosphorylated, growth-suppressive form of the retinoblastoma protein and induction of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Reversal of these cell cycle regulatory effects was coincident with activator-induced down-regulation of PKC alpha, delta, and epsilon. Differential down-regulation of individual PKC isozymes revealed that PKC alpha in particular is sufficient to mediate cell cycle arrest by PKC agonists in this system. Taken together, the data implicate PKC alpha in negative regulation of intestinal epithelial cell growth both in vitro and in situ via pathways which involve modulation of Cip/Kip family cyclin-dependent kinase inhibitors and the retinoblastoma growth suppressor protein.

Glycolytic enzyme binding during entrance to daily torpor in deer mice (Peromyscus maniculatus).

Associations of glycolytic enzymes with the subcellular particulate fraction of skeletal muscle and heart were examined during entrance to daily torpor in deer mice (Peromyscus maniculatus). In skeletal muscle a significant decrease in enzyme binding occurred during torpor entrance for phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase, with an additional significant decrease for phosphofructokinase and pyruvate kinase during the deepest state of torpor. Reductions in enzyme binding during torpor entrance also occurred in heart; significant changes were observed in hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase binding. Contrary to the finding of additional reductions in enzyme binding seen in skeletal muscle, significant increases in enzyme binding during the deepest torpor state were observed for hexokinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase in heart. These results suggest that a decrease in the binding of glycolytic enzymes to subcellular structures in skeletal muscle and heart may be at least partially responsible for initiating the reduction in metabolic rate during torpor entrance. This decreased binding may continue to mediate the metabolic reduction in skeletal muscle throughout torpor; heart, however, may require the use of different molecular mechanisms. The increased binding in heart during the deepest state of torpor may represent an anticipatory response in preparation for increased activity during arousal.

Protein Kinase C-ε Is Necessary for Erythropoietin's Up-regulation of c-myc and for Factor-dependent DNA Synthesis: EVIDENCE FOR DISCRETE SIGNALS FOR GROWTH AND DIFFERENTIATION

Erythropoietin regulates the transcription of the protooncogenes c-myc and c-myb by discrete protein kinase C (PKC)-dependent and protein serine/threonine phosphatase-dependent pathways, respectively (Spangler, R., Bailey, S. C., and Sytkowski, A. J. (1991) J. Biol. Chem. 266, 681-684; Patel H. R, Choi H.-S, and Sytkowski A. J. (1992) J. Biol. Chem. 267, 21300-21302). In the present study we demonstrate that up-regulation of c-myc requires the PKC-epsilon isoform and that this pathway is required for erythropoietin-induced DNA synthesis (growth) but apparently not for beta-globin expression (differentiation). Treatment of Rauscher murine erythroleukemia cells resulted in phosphorylation of phospholipase C-gamma1 and activation of PKC-epsilon as evidenced by its translocation from soluble to particulate subcellular fractions. Artificial down-regulation of PKC-epsilon with antisense oligodeoxynucleotides blocked erythropoietin's up-regulation of c-myc in a concentration-dependent manner. In contrast, antisense oligodeoxynucleotides to PKC-alpha, -beta, -gamma, -delta, and -zeta had no effect. Although down-regulation of PKC-epsilon blocked the increase in c-myc expression, it did not inhibit erythropoietin induction of beta-globin expression, a marker of erythroid differentiation. However, down-regulation of PKC-epsilon did block factor-dependent DNA synthesis quantified by measurement of [3H]thymidine incorporation into newly synthesized DNA of normal murine erythroid cells. The results are consistent with discrete intracellular signals regulating erythroid cell growth and differentiation.

Endothelial Nitric Oxide Synthase Targeting to Caveolae: SPECIFIC INTERACTIONS WITH CAVEOLIN ISOFORMS IN CARDIAC MYOCYTES AND ENDOTHELIAL CELLS

The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae.

The Role of Carboxyl-terminal Basic Amino Acids in Gqα-dependent Activation, Particulate Association, and Nuclear Localization of Phospholipase C-β1

The phospholipase C (PLC)-beta isozymes differ from the PLC-gamma and PLC-delta isozymes in that they possess a long COOH-terminal sequence downstream of their catalytic domain, are activated by alpha subunits of the Gq class of G proteins, associate with the particulate subcellular fraction, and are present in the nucleus. Most of the COOH-terminal domain of PLC-beta isozymes is predicted to be helical, and three regions in this domain, PLC-beta1 residues 911-928 (region 1), 1055-1072 (region 2), and 1109-1126 (region 3), contain a high proportion of basic residues that are highly conserved. Projection of the sequences of these three regions in helical wheels reveals clustering of the basic residues. The role of the COOH terminus and the clustered basic residues in PLC-beta1 was investigated by either truncating the entire COOH-terminal domain (mutant DeltaC) or replacing two or three clustered basic residues with isoleucine (or methionine), and expressing the mutant enzymes in CV-1, Rat-2, or Swiss 3T3 cells. The DeltaC mutant no longer showed the ability to be activated by Gqalpha, to translocate to the nucleus, or to associate with the particulate fraction. Substitution of clusters of basic residues in regions 1 and 2 generally reduced the extent of activation by Gqalpha, whereas substitution of a basic cluster in region 3 had no effect. Substitution of the cluster of lysine residues 914, 921, and 925 in region 1 had the most marked effect, reducing Gqalpha-dependent activity to 10% of that of wild type. All substitution mutants, with the exception of that in which lysine residues 1056, 1063, and 1070 in region 2 were substituted with isoleucine, behaved like the wild-type enzyme in showing an approximately equal distribution between cytoplasm and nucleus; only 12% of the region 2 mutant was present in the nucleus. None of the basic clusters appeared critical for particulate association; however, replacement of each cluster reduced the amount of PLC-beta1 in the particulate fraction by some extent, suggesting that all the basic residues contribute to the association, presumably by interacting with acidic residues in the particulate fraction. Membrane localization of PLC-beta isozymes is therefore likely mediated by both the COOH-terminal domain and the pleckstrin homology domain, the latter of which is known to bind phosphatidylinositol 4,5-biphosphate.

Expression of HIV-1 Envelope Glycoprotein Alters Cellular Calmodulin

Removal of parts of a known calmodulin binding site at the C-terminus of HIV-1 envelope glycoprotein, gp160, can result in diminished infectivity. We investigated whether expression of full length gp160 would result in changes in intracellular calmodulin compared to expression of gp160 truncated to remove both known calmodulin binding sites. Both Western and Northern blots demonstrated that expression of gp160 led to increased calmodulin when compared to expression of truncated gp160. The induced calmodulin was associated preferentially with a particulate subcellular fraction. Confocal immunomicroscopy confirmed the increase in calmodulin and also showed that there was enhanced colocalization of calmodulin with gp160. Understanding of the role of calmodulin in the viral life-cycle may lead to new therapeutics.

The signal transduction pathway of erythropoietin involves three forms of mitogen-activated protein (MAP) kinase in UT7 erythroleukemia cells.

The survival and proliferation of the UT-7 human leukemic cell line is strictly dependent on the presence of either interleukin 3, granulocyte-macrophage colony-stimulating factor or erythropoietin. In these cells, erythropoietin stimulation led to the rapid phosphorylation of several proteins including the erythropoietin receptor and proteins with molecular masses around 45 kDa which could be mitogen-activated protein (MAP) kinases. Separation of cytosol from resting or erythropoietin-stimulated UT-7 cells by anion-exchange chromatography revealed two peaks of myelin basic protein kinase activity. The kinase activity of the first peak was independent of erythropoietin treatment of the cells and corresponded to an unidentified 50-kDa kinase, whereas the second peak was only present in erythropoietin-stimulated cells and corresponded to three forms of MAP kinases with molecular masses of 45, 44 and 42 kDa. The three forms were separated by hydrophobic chromatography and were shown to be activated in erythropoietin-stimulated cells. The 44-kDa and 42-kDa forms corresponded to extracellular signal-regulated kinase (ERK)-1 and ERK-2, respectively. Evidence was obtained showing that the 45-kDa form is not a shifted form of ERK-1 but corresponded to a less well defined form of MAP kinase which may be the previously described ERK-4. MAP kinase activation was detected after 1 min erythropoietin stimulation and remained detectable after more than 1 hour. A role for MAP kinase activation in erythropoietin-stimulated cell proliferation was suggested by the simultaneous inhibition of erythropoietin-induced MAP kinase stimulation and cell proliferation. The potential activator of MAP kinase, RAF-1, was hyperphosphorylated in erythropoietin-stimulated cells and its autophosphorylation activity was strongly increased. The protein adaptor Shc was heavily phosphorylated in UT-7 erythropoietin-stimulated cells and associated strongly with a unidentified 145-kDa protein. However, Shc bound poorly to the activated erythropoietin receptor and most Shc proteins were cytosolic in both unstimulated and erythropoietin-stimulated cells. In contrast, Grb2 associated efficiently with the activated erythropoietin receptor and a significant part of Grb2 was associated to a particulate subcellular fraction upon erythropoietin stimulation.

Expression of constitutive endothelial nitric oxide synthase in human blood platelets

Nitric oxide (NO) activates the soluble isoform of guanylate cyclase in platelets and inhibits platelet function. Several studies suggest the existence of a pathway for NO synthesis in platelets as a form of feedback inhibition, but the identity of the NO synthase (NOS) isoform present within platelets is unknown. We isolated human platelets and synthesized cDNA from platelet RNA for analysis by PCR. Primers for human neuronal or inducible NOS failed to yield a PCR signal. However, primers specific for endothelial NOS (ecNOS) amplified a DNA band of the expected size. Analysis of nucleotide sequence revealed that the amplified DNA is ecNOS. NOS enzyme activity was detected in the platelet particulate subcellular fraction, as previously demonstrated for ecNOS in other cells. Thus, ecNOS is present in human platelets and may play a role in the regulation of platelet function by an endogenous NO pathway.

Dietary lipid and iron modify normal colonic mucosa without affecting phospholipase A2 activity.

Phospholipase A2 (PLA2) functions as the rate-limiting step in arachidonic acid metabolism and in the removal of damaged or peroxidized membrane lipids. It is elevated in some human tumors and may be involved with mechanisms of tumor promotion. In vitro systems have shown PLA2 activity to be altered by variations in fatty acid and antioxidant components. This study encompassed two objectives. First, PLA2, activity in colon tumors produced using azoxymethane (AOM) in Fischer-344 rats was examined. Secondly, this study tested the effect of iron supplementation as a potential pro-oxidant in diets of varied fatty acid composition on PLA2 activity. Diets included 35 or 140 mg/kg or iron in AIN-76A based diets high in corn oil, menhaden oil, or beef tallow. Results for the first objective showed PLA2 activity to be significantly higher in colon tumors than in normal mucosa with the increase due primarily to an increase in activity within a particulate subcellular fraction. In the second objective, fatty acid composition of colon mucosa was altered by both dietary fat and iron. Animals fed beef tallow had the highest level of oleic acid and corn oil-fed animals had the highest level of linoleic acid. Animals fed menhaden oil had the lowest level of arachidonic acid and highest level of alpha-linolenic, eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids. Iron supplementation in diets high in corn oil resulted in decreased membrane composition of palmitoleic and delta-linolenic acid. In spite of these changes in membrane composition, there were no changes in PLA2 activity. These results show that PLA2 activity is increased in AOM-induced tumors but that diet alone does not influence PLA2 activity in this model.

Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.