Gene/Protein Disease Symptom Drug Enzyme Compound
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The present knowledge of glutathione (GSH) peroxidase is briefly reviewed: GSH peroxidase has a molecular weight of about 85,000, consists of four apparently-identical subunits and contains four g atom of selenium/mol. The enzyme-bound selenium can undergo a substrate-induced redox change and is obviously essential for activity. In accordance with the assumption that a selenol group is reversibly oxidized during catalysis, ping-pong kinetics are observed. Limiting maximum velocities and Michaelis constants, indicating the formation of an enzyme-substrate complex, are not detectable. The enzyme is highly specific for GSH but reacts with many hydroperoxides. It can be deduced from the kinetic analysis of GSH peroxidase that in physiological conditions removal of hydroperoxide is largely independent of fluctuations in the cellular concentration of GSH. However, the system will abruptly collapse if the rate of hydroperoxide formation exceeds that of regeneration of GSH. By these considerations, the pathophysiological manifestation of disorders in GSH metabolism and pentose-phosphate shunt may be explained. With regard to its low specificity for hydroperoxides, GSH peroxidase could be involved in various metabolic events such as H2O2 removal in compartments low in catalase, hydroperoxide-mediated mutagenesis, protection of unsaturated lipids in biomembranes, prostaglandin biosynthesis, and regulation of prostacyclin formation.
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PMID:Glutathione peroxidase: fact and fiction. 38 23

Many hormones and drugs exert their effects on cells by increasing cytosolic Ca2+ (Cai2+) and activating protein kinase C (PKC). Each of these actions results in cholestasis in the isolated perfused rat liver, but the responsible mechanisms are unclear. We used isolated rat hepatocyte couplets to observe the direct effects of increased Cai2+ and PKC activation on permeability of the hepatocyte tight junction and canalicular volume, two possible determinants of hepatocyte bile secretion. Couplets were stimulated with the Ca2+ agonist vasopressin (10(-8) M) in the absence and presence of the Ca2+ influx antagonist Ni2+ (5 x 10(-3) M) or with the PKC activator phorbol dibutyrate (10(-6) M). Cai2+ was determined by ratio microspectrofluorometry of indo-1, permeability of the couplet tight junctions was assessed by exclusion of horseradish peroxidase from the canalicular space, and changes in canalicular volume over time were measured directly by optical planimetry. Canalicular volume increased by 1.6 +/- 2.5%/min (mean +/- SD) under basal conditions. In response to vasopressin, there was a rapid 15-fold increase in Cai2+, followed first by an increase in paracellular permeability, then by canalicular collapse (15.9 +/- 5.9%/min). Pretreatment with Ni2+ markedly decreased the vasopressin-induced increase in Cai2+ and abolished both the increase in paracellular permeability and the canalicular collapse. Phorbol dibutyrate also increased paracellular permeability but resulted in neither increased Cai2+ nor canalicular collapse. The PKC inhibitor H-7 reversed the effects of both vasopressin and phorbol dibutyrate on tight junction permeability. Bile secretory pressure, measured in isolated perfused rat liver preparations, was acutely increased by vasopressin, but the increase was augmented rather than inhibited by Ni2+.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hormonal regulation of paracellular permeability in isolated rat hepatocyte couplets. 161 38

The role of cytokeratin filaments in the function of hepatocytes was investigated using a nickel-treated hepatocyte in vitro model. Cytokeratin intermediate filaments were selectively dissociated from the cell cortex by nickel treatment. Cytokeratins and ubiquitin were observed using immunofluorescence and immunoelectron microscopy. Hepatocytic function was assessed by visualizing uptake, transhepatic transport and secretion of fluorescein diacetate and horseradish peroxidase into the bile canaliculi. In control primary cultures, most of the bile canaliculi were surrounded by an inner layer of actin filaments and an outer pericanalicular sheath of cytokeratin filaments and microtubules. The cytoplasmic distribution of ubiquitin was diffuse and particulate. After treatment with NiCl2 (150 micrograms/ml) for 24 hr, the cytokeratin filaments and desmoplakin became focally detached from the cell cortex and retracted to form an aggregate around the nucleus. These aggregates were associated with intense ubiquitin immunoreactivity. Only a few attachments of the cytokeratin filaments to the cell cortex remained. F-actin remained attached to the cell cortex in the areas where the cytokeratin filaments had become detached. The pericanalicular sheath of cytokeratin filaments and the bile canaliculi disappeared and actin was dispersed over the entire cell periphery. Fluorescein diacetate secretion and horseradish peroxidase uptake were almost completely absent in the hepatocytes treated with nickel. The effects of nickel persisted 24 hr after its removal from the medium. It is concluded that cytokeratin intermediate filaments play a critical role in the formation of the bile canaliculus, secretion of fluorescein diacetate and uptake of horseradish peroxidase. Further, our study indicates that cytokeratin ubiquitination occurs during collapse and aggregation of the cytokeratin filaments. The formation of cytokeratin-ubiquitin conjugates during aggregation suggests a role of ubiquitin in the control of cytokeratin organization in hepatocytes in the response to cell stress.
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PMID:Role of cytokeratin intermediate filaments in transhepatic transport and canalicular secretion. 169 Jan 70

The reaction of horseradish peroxidase with alkylhydrazines results in delta-meso-alkylation of the prosthetic heme group and enzyme inactivation (Ator, M. A., David, S. K., and Ortiz de Montellano, P. R. (1987) J. Biol. Chem. 262, 14954-14960). As reported here, enzyme inactivation is associated with the accumulation of intermediates that absorb at approximately 835 nm. The properties of these intermediates, including their collapse to give meso-alkylhemes, identify them as isoporphyrins. The t1/2 values for inactivation and formation of the isoporphyrin intermediate at 25 degrees C are, respectively, 11.6 and 12.5 min for methylhydrazine (2.0 mM), 8.7 and 7.2 min for ethylhydrazine (1.0 mM), and 30 and 25 s for phenylethylhydrazine (50 microM). The isoporphyrin intermediates are surprisingly long-lived, with half-lives (35 degrees C, pH 7.0) of 9, 28, 96, and 450 min for, respectively, the phenylethyl, methyl, n-butyl, and ethyl analogues. pH studies show that protonation of a group with pKa = 5.0-6.5 accelerates isoporphyrin decay and decreases steady state isoporphyrin accumulation. Horseradish peroxidase reconstituted with delta-meso-methylheme, unlike horseradish peroxidase with a heme that has a larger meso-substituent, is catalytically active but is more sensitive to H2O2-mediated degradation of the prosthetic group than is the native enzyme. The delta-meso-methylheme prosthetic group is converted in the reaction with H2O2 to a biliverdin-like product. The results implicate highly stabilized isoporphyrin intermediates in the inactivation of horseradish peroxidase by alkylhydrazines and indicate that inactivation by the meso-alkyl groups is due to steric interference with electron delivery to the heme edge rather than to intrinsic electronic consequences of meso-alkylation. The structural features that stabilize the cationic isoporphyrins may also be involved in stabilization of the Compound I porphyrin radical cation.
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PMID:Stabilized isoporphyrin intermediates in the inactivation of horseradish peroxidase by alkylhydrazines. 272 29

Ligand binding to cell surface receptors induces rapid internalization of ligand-receptor complexes by receptor mediated endocytosis. We have examined the intracellular movement of endocytic vesicles, induced by the lectin concanavalin A (Con A), in cultured rat ovarian granulosa cells using fluorescence and electron microscopy. Within 20 minutes of ligand treatment at 37 degrees C, numerous Con A-containing endocytic vesicles form, which migrate to the cell center by 60 minutes. Double label fluorescence microscopy, using fluorescein-Con-A and rhodamine immunofluorescent staining of tubulin or vimentin, indicates that during vesicle migration microtubules and 10-nm filaments are altered in their organization. By 30 minutes, vesicles are associated with microtubule bundles, which subsequently collapse around the nucleus. Similarly, 10-nm filaments accumulate around the nucleus in conjunction with the perinuclear aggregation of endocytic vesicles. Electron microscopy of Con A-horseradish peroxidase-labeled cells demonstrates that endocytic vesicles fuse to form large receptosome-like structures during intracellular migration and these structures are associated with cytoplasmic microtubules and 10-nm filaments. Taxol, a drug that stabilizes microtubules, prevents endocytic vesicle translocation to the Golgi region. Nocodazole, which causes microtubule disassembly, results in the collapse of 10-nm filaments and the central aggregation of endocytic vesicles. The data indicate that the cytoskeleton participates in the directed intracellular movement of endocytic vesicles; the possible subcellular basis for this movement is discussed.
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PMID:The intracellular movement of endocytic vesicles in cultured granulosa cells. 613 82

Thioglycollate-elicited mouse peritoneal macrophages were cultivated in vitro and endocytosis of native and cationized horseradish peroxidase was studied electron microscopically and biochemically. Native peroxidase (HRP) was ingested by fluid-phase endocytosis and accumulated in lysosomes. Cationized peroxidase (CHRP) bound diffusely to the macrophage surface in a saturable manner. It was then internalized via membrane folds and transferred not only to lysosomes but also to the Golgi complex, mainly those parts referred to as GERL and positive for acid phosphatase activity. Following initial uptake, surface staining for CHRP was lost, although the tracer remained present in the medium. This indicates that anionic binding sites were internalized together with the ligand and not immediately replaced. Accordingly, continued uptake of CHRP occurred at a rate similar to that for HRP. Exposure of the macrophages to cationized ferritin (CF) decreased their ability to bind CHRP. However, after 2 to 4 h in CF-free medium, the CHRP-binding ability returned and raised to 2 to 3 times higher values than in cells not exposed to CF. Treatment with cycloheximide at a concentration that effectively inhibits protein synthesis did not clearly affect this regeneration. These findings support the concept of recirculation of plasma membrane constituents. They further suggest that there exists an intracellular membrane pool which rapidly exchanges with the cell surface. Colchicine removed cytoplasmic microtubules, caused a characteristic disorganization of the Golgi complex, and inhibited uptake of both HRP and CHRP. Additionally, no transport of CHRP to the Golgi complex or GERL was observed in colchicine-treated cells. The regeneration of surface anions after exposure to CF was also delayed. Contrarily, lumicolchicine was without effect on cell morphology and uptake as well as intracellular transport of the tracers. Nevertheless, the effects of colchicine on endocytosis were not necessarily due to a direct role of microtubules. They could be secondary to a disturbed function of the Golgi complex or other organelles after collapse of the microtubular cytoskeleton.
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PMID:Endocytosis of native and cationized horseradish peroxidase by cultured mouse peritoneal macrophages. Variations in cell surface binding and intracellular traffic and effects of colchicine. 733 93

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-hsp 70 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 degrees C heat shock.
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PMID:A heat-shock-like response with cytoskeletal disruption occurs following hydrostatic pressure in MG-63 osteosarcoma cells. 751 Jan 13

We have studied asteroid bodies (ABs) of multinucleated giant cells (MGCs) in a series of sarcoid and foreign body granulomas with a standard streptavidin-biotin peroxidase technique, using commercial antibodies against collagen, vimentin and tubulin on routinely processed tissue as well as, in one case, on fresh frozen sections (FS). Our findings clearly indicate that ABs are products of the microtubule (MT) system and lack collagen. The tubulin in them stains in fresh FS but is "masked" in formalin-fixed tissue. It can be fully "unmasked" by dephosphorylation and partially by trypsinization. Compared to single microtubule organizing centers (MTOCs) in mononuclear cells serving as internal controls, ABs are obvious replicas of centrosome-nucleated MT assemblies from which they differ principally by the disproportionate size of their components and by the invariable vacuolation of the surrounding cytoplasm. Relying on bits of relevant information gleaned from the literature, our observations support the following preliminary conclusions: 1) spokes are massive bundles of MTs rich in tyrosinated alpha-tubulin coassembled in phosphorylated linkages with yet unidentified microtubule associated proteins (MAPs) and probably microfilament proteins; cores are masses of pericentriolar material including amorphous tubulins, MAPs, phosphoproteins and phospholipids; 2) their size, at least in some ABs, appears to indicate the presence of overlapping centrosome-nucleated MTOCs which in monocyte-derived MGCs are known to be multiple; 3) the cytoplasmic vacuolations around them reflect a collapse and retraction of intermediate filaments (IFs), indicating substantial ongoing MT depolymerization with disruption of MT-IF interactions; 4) ABs are products of unusual MTOC dynamics characterized by simultaneous MT assembly and depolymerization; such a phenomenon, termed "microtubule catastrophe", has been recognized in vitro with centrosome-nucleated MT assemblies under conditions of low tubulin concentrations.
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PMID:Asteroid bodies: products of unusual microtubule dynamics in monocyte-derived giant cells. An immunohistochemical study. 789 35

The protein kinase inhibitor staurosporine inhibited, and often abolished, activity-dependent destaining of frog motor nerve terminals that had been preloaded with the fluorescent dye FM1-43. Staurosporine did not, however, block synaptic transmission; staurosporine treated muscles twitched in response to nerve stimulation, and the amplitudes of evoked end plate potentials were reduced only slightly, and in some cases not at all. The blockade of FM1-43 destaining was not reversed by washing, although treatment with black widow spider venom caused complete destaining. Nerve terminal pretreated with staurosporine could subsequently be stained with FM1-43 (and then destained by black widow spider venom). Thus, staurosporine blocked destaining but not staining of nerve terminals. Staurosporine treatment had little effect on the ultra-structure of resting terminals, the main difference we noted being a somewhat closer packing of synaptic vesicles after exposure to staurosporine. However, staurosporine blocked completely the ultrastructural changes produced by prolonged nerve stimulation, such as depletion of synaptic vesicles, appearance of intraterminal cisternae, and the uptake of horseradish peroxidase. The effects of staurosporine were not mimicked by KN-62, H7, calmidozolium, or trifluoroperazine. These and other observations are consistent with, but do not prove the hypothesis that, after exposure to staurosporine, the exocytotic fusion pore behaved like a valve, letting FM1-43 in, but not out, as if staurosporine interfered with the postexocytotic collapse of synaptic vesicles into the surface membrane.
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PMID:Staurosporine blocks evoked release of FM1-43 but not acetylcholine from frog motor nerve terminals. 861 58

It has recently been demonstrated [Pettus et al., J. Neurotrauma, 11 (1994) 507-522] that moderate traumatic brain injury evokes alterations in axolemmal permeability associated with rapid local compaction of axonal neurofilaments (NF). The current communication fully characterized these local NF changes, while also exploring the possibility of other related cytoskeletal abnormalities. A tracer normally excluded by the intact axolemma (horseradish peroxidase) was administered intrathecally in cats, which were then subjected to moderate/severe fluid percussion brain injury (FPI). After survival times ranging from 5 min to 6 h post-traumatic brain injury (TBI), the animals were perfused and processed for light microscopic (LM) and electron microscopic (EM) visualization of horseradish peroxidase (HRP). HRP-containing axons identified by LM, were investigated by EM in both the sagittal and coronal planes. Electron micrographs were videographically captured, digitized, and analyzed for cytoskeletal distribution. Local alterations in axolemmal permeability to HRP were detected, and consistently linked with distinct cytoskeletal changes. Within 5 min of injury, the injured HRP-containing axons displayed a significant decrease in inter-NF spacing associated with a lack of NF side arm projections. Density analysis proved a significant increase in NF packing in the HRP-containing axons, and further revealed an associated significant decrease in microtubule (MT) density. All ultrastructural changes were seen within 5 min of injury, and persisted unchanged for up to 6 h post-TBI. Collectively, these abnormalities suggest that altered axolemmal permeability triggers a rapid, yet persisting general cytoskeletal change most likely linked to local ionic disregulation. We posit that this local cytoskeletal collapse/alteration marks a site of impaired axonal transport, associated with upstream axoplasmic swelling and eventual axonal detachment.
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PMID:Characterization of a distinct set of intra-axonal ultrastructural changes associated with traumatically induced alteration in axolemmal permeability. 881 44


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