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PubMed1985

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3.

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen nonspecific cytotoxic activity. It is furthermore shown that anti-T3 reagents are able to trigger lytic activity in T cell clones characterized as noncytotoxic antigen-specific proliferating T cells. The data presented indicate that perturbation of T3 can trigger the lytic machinery in cytolytic as well as noncytolytic T cell clones.

Antibodies, Monoclonal↗

PubMed2002

Frequent activation of AKT2 kinase in human pancreatic carcinomas.

Activation of AKT/protein kinase B promotes a variety of biological activities important in tumorigenesis, such as cell survival and cell cycle progression. We previously demonstrated amplification and overexpression of the AKT2 gene in a subset of human pancreatic carcinomas. In this investigation, we assessed AKT2 catalytic activity in 50 frozen pancreatic tissues (37 carcinomas, four benign tumors and nine normal pancreata) by in vitro kinase assay. Twelve of 37 (32%) pancreatic carcinomas showed markedly elevated levels of AKT2 activity compared to normal pancreata and begin pancreatic tumors. To delineate mechanisms contributing to AKT2 activation in malignant pancreatic tumors, we examined the status of upstream components of the phosphatilydlinositol 3-kinase (PI3K)/AKT pathway. Western blot analysis revealed loss of PTEN protein expression in two of the 12 pancreatic carcinomas with activated AKT2. In vitro PI3K assays demonstrated high levels of PI3K activity in seven carcinoma specimens that showed AKT2 activation. Immunohistochemical staining confirmed high levels of phosphorylated (active) AKT in malignant pancreatic tumors compared to normal pancreata. Overall, these data suggest that upstream perturbations of the PI3K/AKT pathway contribute to frequent activation of AKT2 in pancreatic cancer, which may contribute to the pathogenesis of this highly aggressive form of human malignancy.

Adenocarcinoma↗

PubMed1995

Mechanical loading stimulates the release of transforming growth factor-beta activity by cultured mouse calvariae and periosteal cells.

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) inhibits bone resorption and stimulates bone formation in cultured fetal mouse calvariae (Klein-Nulend et al., 1986, Arthritis Rheum., 29: 1002-1009). The production of soluble bone factors by such calvariae is also modified (Klein-Nulend et al., 1993, Cell Tissue Res., 271:513-517). Transforming growth factor-beta (TGF-beta) is an important local regulator of bone metabolism and is produced by osteoblasts. In this study, the release of TGF-beta activity as a result of mechanical stress was examined in organ cultures of neonatal mouse calvariae, in primary cultures of calvariae-derived osteoprogenitor (OPR) cells, and in more differentiated osteoblastic (OB) cells. Whole calvariae and calvariae-derived cells were cultured in the presence or absence of IHC for 1-7 days and medium concentrations of active as well as total TGF-beta were measured using a bioassay. IHC (maximum 13 kPa, maximal pressure rate 32.5 kPa/sec) was generated by intermittently (0.3 Hz) compressing the gas phase above the cultures. We found that mechanical loading by IHC stimulated the release of TGF-beta activity from cultured calvariae by twofold after 1 day. IHC also stimulated the release of TGF-beta activity from calvariae-derived cells after 1 and 3 days. The absolute amounts of TGF-beta activity released were lower in OPR cells than in OB cells, but the stimulatory effect of IHC was greater in OPR cells. Total TGF-beta (active and bound) released into the medium was not affected by IHC. IHC did not change the dry weight of the organ cultures, nor the DNA or protein content of the cell cultures. These data show that mechanical perturbation of bone cells, particularly OPR cells, enhances the activation of released TGF-beta. We conclude that modulation of TGF-beta metabolism may be part of the response of bone tissue to mechanical stress.

Animals↗

PubMed1994

The structure of F-actin. Results of global searches using data from electron microscopy and X-ray crystallography.

The structure of F-actin was investigated by fitting the crystallographically determined actin monomer structure to F-actin electron microscopy data sets obtained by a variety of methods. A reciprocal-space global search procedure was applied to non-equatorial reciprocal-space amplitudes and phases of the microscopy data to locate minima. Fits were performed over a range of cross-sectional radii-of-gyration encompassing values obtained from X-ray solution scattering measurements. Five data sets from four laboratories were investigated: one from frozen-hydrated single filaments, three from negatively stained single filaments, and one from negatively stained single-layer paracrystals. In this last case the paracrystal data were straightened to improve resolution. The best fits nearly unanimously favored a monomer orientation having long-pitch connectivity that was close to that obtained by fitting X-ray fiber diffraction patterns. In certain cases where the resolving power was low, competitive fits were obtained with a quite different orientation, one having only protomer connectivity along the genetic helix. Using a running 10-residue deletion from the monomer in the Holmes-type orientation, subtle differences between the monomer structure and the protomer structure in F-actin could be detected. In particular, differences in the "DNase loop" (residues 41 to 50) and the "hydrophobic loop" (residues 264 to 273) were seen in single-filament data. In addition, a perturbation of the structure was seen in a region near residues 81 to 90. A rearrangement within the protomer structure reported in the literature did not produce fits as good as those obtained when using the undistorted monomer crystallographic structure without 10-residue deletions. These results, taken as a whole, provide strong support for a structure of Mg(2+)-ADP F-actin similar to that originally suggested by Holmes et al. but with alterations of the hydrophobic loop, the DNase loop and in the region near residues 81 to 90. These latter two regions have been proposed as secondary binding sites for myosin heads. The available evidence from electron microscopy and from other sources suggests that residues 40 to 49 are disordered in Mg(2+)-ADP F-actin.

Actins↗

PubMed2007

The role of membrane lipids in the induction of macrophage apoptosis by microparticles.

Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular cross-talk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the pre-incubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the fact that in clinical situations with excessive cell death such as malignancies, autoimmune diseases and following chemotherapies high levels of circulating microparticles might modulate phagocytosing cells, a suppression of the immune response might occur due to loss of macrophages.

Animals↗

PubMed1988

Ethionine-induced alterations of enzymes involved in lipid metabolism and their possible relationship to induction of fatty liver.

Changes of enzymes involved in the hepatic metabolism of long-chain fatty acids (palmitoyl-CoA synthetase (EC 6.2.1.3), carnitine palmitoyltransferase (EC 6.2.1.3), glycerophosphate acyltransferase (EC 2.3.1.15)) in the liver of male rats were examined after ethionine exposure. Ethionine administration resulted in a dose- and time-dependent enhancement of the palmitoyl-CoA synthetase activity both in the mitochondrial, peroxisomal and microsomal fractions. The total carnitine palmitoyltransferase activity in the mitochondrial fraction was enhanced. Ethionine administration was also associated with dose- and time-dependent changes of the microsomal glycerophosphate acyltransferase activity, whereas the mitochondrial enzyme activity was marginally affected. The hepatic triacylglycerol content of the ethionine-treated animals was increased. Hepatic lipids were accumulated in large droplets. Serum triacylglycerol and cholesterol were decreased. In particular, the serum HDL-cholesterol level was lowered. The concentration of ATP in the liver decreased. Accumulation of the metabolic product S-adenosylethionine (AdoEth) was observed for the first 2 days of exposure followed by a fall in S-adenosylmethionine (Ado-Met) during the next 10 days. Linear regression analysis of ATP content versus AdoEth and AdoMet showed highly significant correlations. A significant correlation between the hepatic triacylglycerol and AdoEth content was also observed upon ethionine treatment. The data show that ethionine perturbs the hepatic lipid metabolism. Enhanced esterification of long-chain fatty acids, but not a simple reduction of their oxidation, might contribute to ethionine-induced fatty liver in addition to a block in secretion of lipoproteins and decreased protein synthesis.

Acyltransferases↗

PubMed1991

NMR studies of fluorinated visual pigment analogs.

The 19F-nmr chemical shift data of isomeric pigments (11-cis and 9-cis) of four vinyl fluororhodopsins and two trifluororhodopsins have been recorded. When compared with model protonated Schiff bases, a set of F-nmr opsin shift parameter (FOS) was obtained. The data revealed regiospecific protein perturbations on the F-resonances. They can be interpreted in terms of specific protein interactions such as the postulated second point charge and other polar interactions as well as the common hydrophobic protein perturbation.

Fluorine↗

PubMed1987

Effect of ethanol on amylase secretion and cellular calcium homeostasis in pancreatic acini from normal and ethanol-fed rats.

The effects of ethanol on stimulus-secretion coupling were assessed by studying amylase release, Ca2+-homeostasis, and changes in physical properties of membranes in isolated rat pancreatic acini. In acini from normal rats, ethanol (50 mM and above) in vitro caused a dose-dependent stimulation of amylase release and an increase in cytosolic free Ca2+ concentration. Ethanol did not affect amylase secretion stimulated by cholecystokinin-octapeptide (CCK8), a secretagogue that acts by increasing cytosolic free Ca2+ levels, but did potentiate the secretion of amylase induced by vasoactive intestinal peptide (VIP) which raises intracellular cAMP. Ethanol also increased the rate of 45Ca2+ exchange. In acini labeled with the spin-probe 12-doxyl stearic acid, ethanol disordered the pancreatic plasma membranes. By contrast, in acini from animals that had chronically (6-7 weeks) ingested ethanol, the membranes were resistant to this disordering effect of ethanol. Chronic ethanol feeding lowered the total cellular calcium content and ionophore (A23187)-releasable pools of acinar calcium (11 and 24% respectively), and led to a 15-30% decrease in the rate of 45Ca2+ exchange. Chronic ethanol ingestion also lowered the basal rate of amylase secretion, but ethanol in vitro stimulated amylase secretion more than in control preparations. However, these differences in basal and ethanol-induced amylase secretion were not accompanied by corresponding changes in intracellular free Ca2+. The data suggest that ethanol perturbs cell membranes and also disturbs cellular Ca2+ homeostasis. These effects may explain its actions as a weak Ca2+-mediated secretagogue. However, the membrane alterations induced by chronic ethanol feeding do not prevent the ethanol-induced interference with cellular calcium homeostasis.

Amylases↗

PubMed1988

Link-Vertex Analysis of Purkinje cell dendritic trees from the murine cerebellum.

A method of analysis of dendritic trees is described, called Link-Vertex Analysis, which defines the branching patterns (topology) by means of a discontinuous function, the terminal/link (T/L) vertex ratio, computed at all successive vertex orders within a network, and represented as a graphical plot for a given tree. The T/L vertex ratio is defined as the number (n) of terminal (pendant) vertices (Vp) divided by the total number (n) of all link vertices (both dichotomous, Vd, and trichotomous, Vt) multiplied by the number of segments arising from each vertex--i.e., nVp/(2nVd + 3nVt) per vertex order. Hypotheses of growth are simulated by computer modelling and the distributions of computer generated T/L vertex ratios per order compared with those of actual dendritic trees. The differential distribution of T/L vertex ratios per order in trees grown by either random terminal or random segmental branching discriminates between these two forms of growth. Significant advances of this method include the detection of regional variations in growth and remodelling in a tree and the incorporation of Vt with little perturbation of the Vd data. The analytical powers of this method have been tested using the dendritic fields of 20-day-old mouse cerebellar Purkinje cells defining possible temporal sequences and relative contributions of random and non-random terminal growth, and also the frequency of branching over a growth front relative to that of the rest of the tree.

Animals↗

PubMed1985

Inhibition of macrophage activation by calcium channel blockers and calmodulin antagonists.

The biochemical mechanisms by which macrophages become activated to the tumoricidal state are poorly understood. To investigate the role of calcium in this process, the effect of calcium channel blockers and calmodulin antagonists on the acquisition of tumoricidal properties by macrophages activated by a number of different agents was examined. Activation of thioglycollate-stimulated C57BL/6 mouse peritoneal macrophages by macrophage activation factor (MAF) plus LPS, IFN-gamma plus LPS or the calcium ionophore, A23187, was inhibited in a dose-dependent fashion by the calcium channel blockers nifedipine and verapamil. These agents blocked the influx of 45Ca into macrophages activated by MAF plus LPS. Macrophage activation was also inhibited by chlorpromazine, W-7, and calmidazolium at concentrations known to perturb calmodulin function. The data suggest that activation of macrophages to the tumoricidal state is a calcium-dependent process involving the participation of calcium-regulated biochemical reactions whose activities can be modulated by pharmacological agents that frustrate transmembrane calcium fluxes and/or inhibit calmodulin function.

Animals↗

PubMed1994

Pulmonary lung surfactant synthetic peptide concentration-dependent modulation of DPPC and POPG acyl chain order in a DPPC:POPG:palmitic acid lipid mixture.

Lung surfactant-associated protein interaction with lipid matrices and the effects on lipid thermotropic phase behavior are areas of active research. Many studies limit the lipids to a single or two-component system. The current investigation utilizes a three-lipid component matrix (DPPC:POPG:palmitic acid) to investigate the impact of a synthetic surfactant protein B fragment (SP-B 53-78 DiACM) on the dynamic surface activity of the lipid admixture as measured by a Wilhelmy surface balance. Also, the modulation of the individual lipid acyl chain order by the peptide within the lipid matrix is studied through the use of thermal perturbation FTIR spectroscopy. The data clearly demonstrate a concentration-dependent effect of the peptide on the surface activity with an improvement in the dynamic surface tension diagram characteristics (decreased surface tension and increased collapse plateau) especially at low, 0.36 M%, peptide concentrations. These effects are diminished upon further addition of the peptide. FTIR spectral data demonstrate that the peptide addition results in a significant increase in the acyl chain order of the DPPC and POPG components as measured by the position of the methylene stretching vibrational bands. DPPC is most sensitive to the peptide presence, while the palmitic acid is least affected. The transition temperatures of the individual lipids are also increased with the addition of the peptide. The presence of POPG in the matrix achieves the surface activity similarly seen with natural lung surfactant relative to a DPPC/palmitic acid lipid matrix alone. Its presence increases the sensitivity of the DPPC acyl chains to the presence of the peptide. These effects on the chain order are most probably related to the increased acyl chain fluidity which POPG imparts to the lipid matrix because of the presence of the cis double bond. The phosphatidylglycerol headgroup also adds a negative charge to the lipid matrix which enhances the peptide-lipid interaction. Although the palmitic acid is minimally affected by the peptide, its presence, as suggested by surface balance measurements, results in the establishment of a stable lipid film with DPPC, capable of achieving low surface tension values.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Multiresolution decomposition of non-stationary EEG signals: a preliminary study.

Wavelet representation is a recent development in the analysis of non-stationary signals. Its possibilities for use in the description of time-frequency characteristics of both transients in spontaneous EEG and time-varying rhythms in event related brain activity are explored here. By way of illustration, multiresolution decompositions of a wide variety of EEG transients are carried out in this work, including spike-and-waves, single spikes, sharp waves, blink artifacts, frontal intermittent rhythmic delta activity (FIRDA) and paroxysmal delta activity. Also, the application of the wavelet representation to study related spectra perturbations is illustrated with data from psychophysical experiments on the perception of image motion. The results demonstrate the capabilities of the wavelet transform, as an alternative to the Fourier transform, for the representation and analysis of non-stationary EEG signals.

Artifacts↗

PubMed1982

Volume of distribution and transcapillary passage of small unilamellar vesicles.

This study investigated the biodistribution of bovine brain sphingomyelin (SM)/cholesterol (CH) (2/1; M/M) small unilamellar vesicles (SUV) in mice, addressing specifically the volume of distribution and transcapillary passage of the SUV. The complex of nitrilotriacetic acid with In-111 or Ga-67 ions was encapsulated in the SUV as the radioactive marker for various studies. The structural integrity of liposomes in vitro and in vivo was monitored by the technique of gamma ray perturbed angular correlation. Our data suggested that initially the SM/CH SUV remained within the vascular system and occupied a volume of distribution approximately 1.28 times larger than that of erythrocytes in the vascular system of mice. However, our data also indicated that with time the SM/CH SUV could get out of the vascular system of mice and were taken up by surrounding tissues over a period of 24 hours.

Animals↗

PubMed1988

Substances that polymerize or depolymerize cytoskeletal proteins affect platelet spreading and thrombus-formation on surfaces coated with human collagen isotypes I, IV, and V.

The effect of substances that affect platelet cytoskeleton on the interaction of gel-filtered platelets with surfaces coated with human monomeric type I, IV, and V collagen was studied. The sulfhydryl group oxidizing agent azodicarboxylic acid-bis-dimethylamide (diamide) which causes disulfide-linked polymer formation of certain cytoskeletal proteins, the actin-polymerization inhibitor, cytochalasin B, and 2-mercaptopropionylglycine (2-MPG), a cell-permeable SH-reagent, completely abolish adhesion-induced platelet spreading and mural platelet aggregate formation on collagen-coated surfaces. Extrusion of pseudopods was inhibited by cytochalasin B and 2-MPG as well as by diamide, but only the latter caused spherulation of platelets, whereas cytochalasin B and 2-MPG left the discoid shape of resting platelets intact. These effects are dose-dependent and are not accounted for by a chemical modification of the collagenous substrates by the cytoskeletal perturbing substances. The present data indicate that (i) cytoskeletal rearrangements are essential in adhesion-induced platelet spreading and aggregate formation on surfaces coated with collagen, but not in supporting the initial attachment of native platelets to the substrate; (ii) both, polymerization and depolymerization of actin filaments affect platelet activation; (iii) the sulfhydryl-disulfide status of the platelet seems to be a possible target for anti-platelet drugs, since chemical modification of platelets by the GSH-GSSG-active substances, diamide and 2-MPG, leads to a reversible inhibition of adhesion-induced platelet activation.

Chromatography, Gel↗

PubMed1989

A unified theory of mental illness.

I propose that in certain mental illnesses the mechanics for making, releasing, and responding to neurotransmitters are all normal; what is faulty is a portion of the mechanism whereby the brain recognizes abnormal behavior and feeds back on to the neuronal pathways producing the behavior to alter function of that neuronal system and re-establish normal behavior. All the above uses the physical structure of the brain. The existence of the feedback mechanism, and its perturbation, is deduced from data about the mechanism of antidepressants.

Affect↗

PubMed1988

Effect of acetaminophen hepatotoxicity on hepatic mitochondrial and microsomal calcium contents in mice.

The effect of toxic doses of acetaminophen on hepatic intracellular calcium compartmentation were studied in mice. No effects on the calcium contents of the mitochondria, microsomes or cytosol were observed 4 h after the administration of 175 and 375 mg/kg acetaminophen when compared to saline-treated controls. However, doses of 500 and 750 mg/kg of acetaminophen increased mitochondrial calcium contents at this time. Also, the 750 mg/kg dose caused marked alterations in the calcium contents of microsomal and cytosolic compartments. The time-course of the onset of these effects was examined using a 500 mg/kg dose. No changes in either mitochondrial, microsomal or cytosolic calcium contents were observed in the livers of mice treated with acetaminophen compared to saline-treated controls at either 1 or 2 h after dose administration. However, at 3, 4 and 24 h after acetaminophen, mitochondrial and cytosolic calcium contents were significantly increased above control values. The increases in mitochondrial and cytosolic calcium contents observed in the acetaminophen-intoxicated mouse liver appear to occur at the same time as the appearance of plasma membrane damage, as measured by sorbitol dehydrogenase leakage. The data suggest that a perturbation in hepatic calcium compartmentation is not an early event in acetaminophen-induced hepatotoxicity in the mouse.

Acetaminophen↗

PubMed2005

Effect of oxidative stress on translocation of DAF-16 in oxygen-sensitive mutants, mev-1 and gas-1 of Caenorhabditis elegans.

Mutations in the mev-1 and gas-1 genes of the nematode Caenorhabditis elegans render animals hypersensitive to oxygen and paraquat, and lead to premature aging. We show that both mutants overproduce superoxide anion in isolated sub-mitochondrial particles, which probably explains their hypersensitivity to oxidative stress. The daf-16 gene encodes a fork-head transcription factor that is negatively regulated by an insulin-signaling pathway. In wild-type animals, the DAF-16 protein normally resides in the cytoplasm and only becomes translocated to nuclei upon activating stimuli such as oxidative stress. Conversely, DAF-16 resides constitutively in the nuclei of mev-1 and gas-1 mutants even under normal growth conditions. Supplementation of the antioxidant coenzyme Q(10) reversed this nuclear translocation of DAF-16. Since both gas-1 and mev-1 encode subunits of electron transport chain complexes, these data illustrate how mitochondrial perturbations can impact signal transduction pathways.

Active Transport, Cell Nucleus↗

PubMed2004

Structural model of the UbcH5B/CNOT4 complex revealed by combining NMR, mutagenesis, and docking approaches.

The protein CNOT4 possesses an N-terminal RING finger domain that acts as an E3 ubiquitin ligase and specifically interacts with UbcH5B, a ubiquitin-conjugating enzyme. The structure of the CNOT4 RING domain has been solved and the amino acids important for the binding to UbcH5B have been mapped. Here, the residues of UbcH5B important for the binding to CNOT4 RING domain were identified by NMR chemical shift perturbation experiments, and these data were used to generate structural models of the complex with the program HADDOCK. Together with the NMR data, additional biochemical data were included in a second docking, and comparisons of the resulting model with the structure of the c-Cbl/UbcH7 complex reveal some significant differences, notably at specific residues, and give structural insights into the E2/E3 specificity.

Amino Acid Sequence↗