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K Wu

Publications and source records attributed to K Wu.

At least 289 records · Page 16Linked to original sources
PubMed1988

Neurochemical characteristics of a postsynaptic density fraction isolated from adult canine hippocampus.

Postsynaptic density and synaptic membrane fractions isolated from hippocampal tissue have been compared to those previously isolated from cerebellum and cerebral cortex. In all respects examined, the isolated hippocampal preparations are similar to the cerebral cortex fractions. The morphology of the postsynaptic density (PSD) preparation is the same and the protein composition is similar, but with higher concentrations of the 51-kDa major protein and of calmodulin, and lower concentrations of actin, in the hippocampal PSD fraction. The binding characteristics for glutamate and GABA are also similar between the two fractions, but with higher Bmax and KD glutamate values and lower Bmax and higher KD GABA values for the hippocampal PSD preparation. Both preparations contain GABAA and GABAB receptors. The PSD fraction contains, as does the cerebral cortex fraction, a calmodulin-dependent binding of the Ca2+ channel antagonist, nitrendipine, as well as a cAMP-dependent and a Ca2+/calmodulin-dependent protein kinase, with the same respective substrates. The value of the hippocampal fractions for studies on long-term potentiation and on kindling in the hippocampus is discussed.

Animals↗
PubMed1988

Transsynaptic impulse activity regulates postsynaptic density molecules in developing and adult rat superior cervical ganglion.

Ganglionic postsynaptic density protein (PSDp) was used to monitor the influence of transsynaptic impulse activity on synaptic structure in the developing and adult rat superior cervical sympathetic ganglion (SCG). Since transsynaptic activity is known to regulate ontogeny of postsynaptic transmitter enzymes, we initially studied the developing ganglion. Denervation in neonates prevented normal development, decreasing calmodulin binding to the ganglionic PSDp by 71% after 4 weeks. During this period, denervation elicited only a 42% decrease in total protein of the synaptic membrane fraction, suggesting that innervation regulates development of various synaptic components differentially. Effects of denervation were extremely rapid, resulting in a 44% decrease in calmodulin binding within 1 day, consistent with regulation by a signaling process such as impulse activity. The effect of impulse activity was examined more directly in adults by treatment with the agents reserpine or phenoxybenzamine, which elicit reflex increases in sympathetic transmission. Administration of reserpine resulted in a progressive 90% increase in calmodulin binding to the PSDp over 4 weeks. Phenoxybenzamine also elicited an increase, mimicking the effects of reserpine. Neither agent altered total protein of the synaptic membrane fraction, suggesting that impulse activity regulates specific synaptic components. Finally, ganglionic denervation in adults decreased PSDp binding within 12 hr, consistent with acute effects of impulse reduction. Our results suggest that transsynaptic impulse activity plays an important role in regulation of specific molecular components of the synapse.

Animals↗
PubMed1988

Interleukin 2 receptor expression by macrophages in inflammatory bowel disease.

The expression of interleukin 2 receptor by macrophages from normal and inflamed terminal ileum and colon has been studied by using two monoclonal antibodies. In tissue sections from normal ileum and colon, scattered positive lymphocytes and only occasional weakly positive macrophages were seen. In ileal and colonic Crohn's disease or ulcerative colitis many positive macrophages and lymphocytes were seen in the lamina propria. These findings were confirmed by staining cytospin preparations of isolated intestinal mononuclear cells. The isolated macrophages were able to phagocytose opsonized zymosan and the majority were able to undergo a respiratory burst when triggered with opsonized zymosan or phorbol myristate acetate (PMA), suggesting that they were activated. Stimulation with interferon-gamma or lipopolysaccharide did not increase the number of macrophages staining with the antibodies to the interleukin 2 receptor. Therefore we postulate that a large majority of the macrophages expressing interleukin 2 receptor in inflammatory bowel disease are a recently recruited population of cells.

Antibodies↗
PubMed1987

Interactions of polymerizable phosphatidylcholine vesicles with blood components: relevance to biocompatibility.

We have studied the biocompatibility properties of polymerizable phosphatidylcholine bilayer membranes, in the form of liposomes, with a view toward the eventual utilization of such polymerized lipid assemblies in drug carrier systems or as surface coatings for biomaterials. The SH-based polymerizable lipid 1,2-bis[1,2-(lipoyl)dodecanoyl]-sn-glycero-3-phosphocholine (dilipoyl lipid, DLL) and the methacryl-based lipid 1,2-bis[(methacryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (dipolymerizable lipid, DPL) were studied in comparison to 'conventional' zwitterionic or charged phospholipids. We examined binding of serum proteins to liposomes and effects of liposomes on fibrin clot formation and on platelet aggregation. All types of liposomes tested bound complex mixtures of serum proteins with IgG being the most abundant bound component. DPL vesicles and anionic vesicles bound substantially more protein than other vesicle types. Polymerized DPL vesicles uniquely bound a protein of about 53 kDa which was not bound to other types of phosphatidylcholine liposomes. Likewise polymerized DPL vesicles, but not other types of phosphatidylcholine vesicles, caused a marked alteration in coagulation as measured by activated partial thromboplastin time (APTT) and prothrombin time (PT) tests; this effect was shown to be due to binding and depletion of clothing factor V by the DPL polymerized vesicles. Polymerized DPL liposomes and DLL liposomes in polymerized or nonpolymerized form, were without substantial effect on platelet aggregation. However, DPL nonpolymerized vesicles, while not causing aggregation, did impair ADP-induced aggregation of platelets. These studies suggest that SH based polymerizable lipids of the DLL type may be very suitable for in vivo use in the contexts of drug delivery systems or biomaterials development. Methacryloyl-based lipids of the DPL type seem to display interactions with the hemostatic process which militate against their in vivo utilization.

Biocompatible Materials↗
PubMed1987

Regulation of molecular components of the synapse in the developing and adult rat superior cervical ganglion.

Rat superior cervical sympathetic ganglion was used to begin studying the regulation of molecular components of the synapse. Ganglionic postsynaptic densities (PSDs)exhibited a thin, disc-shaped profile electron microscopically, comparable to that described for brain. Moreover, the presumptive ganglionic PSD protein (PSDp) was phosphorylated in the presence of Ca2+ and calmodulin, bound 125I-labeled calmodulin, and exhibited a Mr of 51,000, all characteristic of the major PSD protein of brain. These initial studies indicated that ganglionic PSDp and the major PSD protein of brain are comparable, allowing us to study synaptic regulation in the well-defined superior cervical sympathetic ganglion. To obtain enough quantities of ganglionic PSDp, we used synaptic membrane fractions. During postnatal development, calmodulin binding to the ganglionic PSDp increased 411-fold per ganglion from birth to 60 days, whereas synaptic membrane protein increased only 4.5-fold. Consequently, different synaptic components apparently develop differently. Moreover, denervation of the superior cervical sympathetic ganglion in adult rats caused an 85% decrease in ganglionic PSDp-calmodulin binding, but denervation caused no change in synaptic membrane protein 2 weeks postoperatively. Our observations suggest that presynaptic innervation selectively regulates specific molecular components of the postsynaptic membrane structure.

Age Factors↗
PubMed1986

Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection.

The mouse inner cell mass is established by cells that are allocated to internal positions after the 8-cell stage. We analyzed the timing of this allocation by microinjecting two cell lineage markers, horseradish peroxidase and rhodamine-conjugated dextran, into mouse blastomeres at the 8- to 32-cell stage. Prospective analysis was performed by coinjection of peroxidase and dextran, followed by 12-22 hr of culture and staining for peroxidase activity; retrospective analysis was performed by injection of peroxidase alone and localization of sister cells without further culture. Both approaches indicated that cells are allocated to internal positions during the fourth and fifth cleavage divisions, but not the sixth cleavage division, of the mouse embryo. Thus, outer cells can have inner descendants until the late morula/early blastocyst (32-cell) stage, but cells remaining outside after the fifth cleavage division are restricted to a trophectoderm fate. This information about cell lineage indicates that the previously observed totipotency of the cleaving mammalian embryo's cells is a regulative attribute that is used in normal development.

Animals↗
PubMed1986

Characteristics of a Ca2+/calmodulin-dependent binding of the Ca2+ channel antagonist, nitrendipine, to a postsynaptic density fraction isolated from canine cerebral cortex.

Synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from the cerebral cortex (CTX) and cerebellum (CL) of the canine brain were found to contain one class of specific nitrendipine binding sites. The specific binding constants were: CTX-SM, Kd = 110 pM (Bmax = 126 fmol/mg protein); CTX-PSD, Kd = 207 pM (Bmax = 196 fmol/mg); CL-SM, Kd = 100 pM (Bmax = 65 fmol/mg); CL-PSD, Kd = 189 pM (Bmax = 80 fmol/mg). Treatment of the CTX-SM and CTX-PSD fractions with 0.5% deoxycholate and 1.0% N-lauroyl sarcosinate removed 88-91% and 42-51% of the nitrendipine binding, respectively, indicating that the major nitrendipine binding present in the SM fractions are of non-synaptic origin. Moreover, the percentages of total protein and specific nitrendipine binding removed from PSDs by these detergents were similar, indicating no preferential dissociation of the latter, and suggesting that the receptor protein is firmly bound and is probably an intrinsic component of the PSD fraction. Both Ca2+ and calmodulin were found to be important for the binding of nitrendipine to the CTX-SM and CTX-PSD fractions since: R24571, a calmodulin antagonist, was found to inhibit nitrendipine binding to the CTX-SM and CTX-PSD fractions with IC50 values of 1.1 microM and 0.9 microM, respectively; removal of Ca2+ from the CTX-SM and CTX-PSD fractions with 0.2 mM EGTA resulted in losses of specific nitrendipine binding of 80 and 90%, respectively; Ca2+ alone restored nitrendipine binding to EGTA-pretreated CTX-SM fractions and not to CTX-PSD fractions, with the latter needing both Ca2+ and calmodulin to restore nitrendipine binding; EGTA treatment removed 14-16% and 89-91% of nitrendipine bound to the CTX-SM and CTX-PSD fractions, respectively, suggesting that calmodulin (but not Ca2+) is needed to maintain the nitrendipine-nitrendipine receptor-calmodulin complex; Ca2+-reconstituted EGTA-pretreated CTX-SM fractions and the Ca2+ plus calmodulin-reconstituted EGTA-pretreated CTX-SM and CTX-PSD fractions were found to have similar binding constants to those for the corresponding native, untreated fractions; and the Ca2+/calmodulin dependency on nitrendipine binding was similar to the well-known Ca2+/calmodulin dependency on phosphorylation in EGTA-pretreated PSD fractions. It needed much less Ca2+ to saturate Ca2+/calmodulin-dependent phosphorylation of the pretreated CTX-PSD fractions than the nitrendipine binding. Yet, less calmodulin was needed to saturate nitrendipine binding than the phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Binding of L-[3H]glutamate to fresh or frozen synaptic membrane and postsynaptic density fractions isolated from cerebral cortex and cerebellum of fresh or frozen canine brain.

Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[3H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the Bmax value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl- increased the number of L-glutamate binding sites and the effect was enhanced by Ca2+; Ca2+ alone had no significant effect; (b) the Cl-/Ca2+-sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing; (c) the effect of Na+ ion was biphasic; low concentration of Na+ (less than 5 mM) decreased Cl-/Ca2+-dependent L-glutamate binding sites, whereas at higher concentrations of Na+ the binding of glutamate was found to increase either in the presence or absence of Ca2+ and Cl-. In addition, the K+ ion (50 mM) was found to decrease the Na+-independent and Cl-/Ca2+-independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na+-dependent and Cl-/Ca2+-independent L-glutamate binding by 93%; in the presence of Cl-/Ca2+, the K+ ion decreased the Na+-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na+-dependent L-glutamate binding sites assayed in the absence of Ca2+ and Cl-. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na+-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na+-dependent nor APB (or Cl-/Ca2+)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminobutyrates↗
PubMed1985

Existence of a Ca2+-dependent K+ channel in synaptic membrane and postsynaptic density fractions isolated from canine cerebral cortex and cerebellum, as determined by apamin binding.

Apamin, a 18-amino acid neurotoxin isolated from bee venom, is a specific blocker of one class of the Ca2+-dependent K+ channels. The monoiodo derivative of the toxin with high specific radioactivity (1600 Ci/mmol) has been used to study its binding to synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brains. The Bmax (30.2 fmol/mg protein) for CTX-PSD is about twice that for CTX-SM (17.3 fmol/mg protein), suggesting a concentration of the apamin receptor protein in CTX-PSD over CTX-SM fractions. The lower value of Bmax for CL-PSD (12.3 fmol/mg protein), and the higher Kd value (51 pM) than for CTX-SM (33 pM), CTX-PSD (24 pM), and CL-SM (39 pM), may reflect the disruptive effect of Triton X-100 on these thin structures. The values of Bmax and Kd for CTX-SM are similar to those (22.0 fmol/mg protein and 33 pM) for rat CTX-SM. Both Ca2+ and Na+ inhibit apamin binding to CTX-PSD with K0.5 values of 14 and 31 mM, respectively, while the optimum concentration of KCl for activation is 5 mM. All these values are similar to those found for rat synaptosomes. Covalent labeling of the apamin binding protein, using the non-cleavable cross-linker, disuccinimidyl suberate, reveals an apamin binding polypeptide of 27 kdaltons under reducing and denaturing conditions in both the CTX-SM and CTX-PSD preparations, similar to that (28 kdaltons) reported for rat CTX-SM fractions. Prior phosphorylation of isolated CTX-PSD had no effect on apamin binding, nor did apamin binding influence subsequent phosphorylation of CTX-PSD. Calmodulin, an intrinsic PSD protein, may not play a role in apamin binding to PSD, since addition of calmodulin, or removal of the calmodulin by EGTA treatment, resulted in no change in the binding capacity of the PSD. The apamin binding protein seems to be bound quite firmly in the CTX-PSD fraction since treatments with 0.5% deoxycholate, 1% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl and 1.0 M KCl, could only remove the apamin-receptor complexes from CTX-PSD by 40, 55, 52, 12 and 15%, respectively. These results contrast with the findings that the two detergents mentioned solubilize 80-93% of the receptor from synaptosomal or synaptic membrane fractions, indicating that a good deal of the receptor in these fractions is membrane-bound and not connected to the PSD.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Developmental and cytogenetic effects of caffeine on mouse blastocysts, alone or in combination with benzo(a)pyrene.

Mouse blastocysts were treated with caffeine and/or benzo(a)pyrene (BP), and the effects on development and on induction of sister chromatid exchanges (SCEs) were examined. Caffeine interfered with blastocyst development in a dose-related manner. At 4 mM, the highest concentration tested, caffeine interfered with development of blastocysts to all four endpoints: hatching, trophoblast outgrowth, inner cell mass (ICM) growth, and two-layer (primary endoderm and ectoderm) differentiation of ICMs. At 2 mM, caffeine reduced the incidence of both ICM growth and differentiation but did not affect hatching or formation of trophoblast outgrowths. At 1 mM, caffeine interfered only with ICM differentiation. Cell proliferation was least sensitive to caffeine and was reduced at concentrations of greater than or equal to 2 mM. Induction of SCEs was most sensitive to caffeine exposure; an increase in SCE frequency was observed at 0.1 and 0.5 mM. When caffeine was added to cultures with BP (1 microM, a concentration that was not embryotoxic and did not induce SCEs), both embryotoxic effects and SCE frequency were increased. The enhancing effect on SCE induction was particularly marked; as little as 0.1 mM caffeine was sufficient to cause doubling of induced SCE frequencies when added to cultures with BP.

Animals↗
PubMed1985

Cytochrome P-450 metabolic activity in embryonic and extraembryonic tissue lineages of mouse embryos.

Mouse morulae, blastocysts, and embryonic and extraembryonic tissue layers were examined for benzo[a]-pyrene metabolism by cytochrome P-450, using the sister chromatid exchange assay. Benzo[a]pyrene exposure in vitro increased sister chromatid exchanges in blastocysts of all genetically responsive mice examined [BALB/cDub, C3H/AnfCum, and outbred Dub:(ICR) strains] but not blastocysts of the nonresponsive AKR/J strain. Benzo[a]pyrene treatment of responsive 7 1/2- and 8 1/2-day (postimplantation-stage) embryos, either intact or as separate tissue layers, increased sister chromatid exchanges in tissues of both embryonic and extraembryonic lineages--i.e., in the embryo proper, in isolated embryonic ectoderm, and in yolk sac, chorion, extraembryonic ectoderm, and extraembryonic endoderm layers. These results indicate that cytochrome P-450 is active in most or all tissues of the early mammalian embryo. It could metabolize xenobiotic molecules reaching the conceptus near the onset of morphogenesis and organogenesis, or it could have another as yet undefined role in normal development.

Age Factors↗
PubMed1985

Neurofibromatosis and malignancy.

Neural crest and nonneural crest tumors occur frequently in neurofibromatosis (NF). We report one case of NF and recurrent malignant fibrous histiocytoma, a tumor that is uncommon in childhood, and another case of the concomitant occurrence of NF, hemophilia B, and a paratesticular rhabdomyosarcoma.

Adolescent↗
PubMed1984

The effect of spicules obtained from sickle red cells on clotting activity.

Spicules from sickle red cells were examined for their effects on the clotting activity of blood. The spicules were obtained from the sickle red cells after deoxygenation and oxygenation and were tested for clotting activity with Russell's viper venom assay. A marked increase in clotting activity was observed when spicules were added to the system. The increase was distinctly greater than that observed after the addition of sickle red cells while normal red cells had little effect. Vesicles prepared from sickle or normal red cells by incubation with the ionophore A-23187+Ca2+ also markedly increased clotting activity. The effect of spicules or vesicles on the clotting system may be related to reorganization of phospholipid in the spectrin-poor membrane of the spicules or vesicles. Because of these effects, the spicules from the sickle red cells may contribute to the hypercoagulable state in these patients and possibly to their vaso-occlusive crises since free spicules are present in their plasma. Vesicles from red cells from other types of anaemia with hypercoagulability may have a similar effect on coagulation.

Anemia, Sickle Cell↗
PubMed1983

Dissociability of enzyme-alpha 2-macroglobulin complexes.

Experiments were performed to measure the extent to which enzymes bound to alpha 2-macroglobulin (alpha 2M) could be dissociated from the complex. Noncovalent complexes are known to exist between alpha 2M and proteases, such as methyl-trypsin that have had their lysyl amino covalently blocked. Complexes between the inhibitor and native enzymes also have a certain fraction noncovalent binding. Because of the severe steric hindrance imposed on enzymes bound to alpha 2M, even in the noncovalent mode, it has been proposed in the literature that they are not dissociable in the usual sense but, rather, are "trapped" in clathrate-like complexes. The results presented here show that lysyl-blocked methyl-thrombin, or native thrombin are released from their alpha 2M complex by an excess of other lysyl-blocked or native proteases. Under conditions where native thrombin is displaced, labeled enzymes can be incorporated, indicating the inhibitor is intact by the criterion of incorporating enzymes. Likewise, native elastase can be released from its alpha 2M complex by excess cold elastase or the inactive anhydrotrypsin, the latter experiment being carried out with an excess of the low-molecular-weight inhibitor diisopropyl phosphofluoridate. In conjunction with previous results showing that lysyl-blocked enzymes are removed from alpha 2M by soybean trypsin inhibitor, the data indicate that, however sterically hindered, alpha 2M-bound enzymes are dissociable and no unique "trapped" intermediate need be postulated.

Binding Sites↗