Dual Activation Mechanism Research Articles

Dual activation in a homolytic coupling reaction promoted by an enantioselective dinuclear vanadium(IV) catalyst

A new concept of dual activation catalysis in homolytic coupling reaction of 2-naphthol derivatives is described. The dinuclear vanadium(IV) catalyst ( R, S, S)- 1a promotes the oxidative coupling reaction of 2-naphthol derivatives with high reactivity and enantioselectivity. This dual activation mechanism is supported by the fact that the reaction rate of oxidative coupling of 2-naphthol promoted by the ( R, S, S)- 1a is 48 times faster than that of the mononuclear complex ( S)- 3 and a lower catalyst loading of ( R, S, S)- 1a shows higher catalyst efficiency both in enantioselectivity and chemical yield.

A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol.

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.

Structural basis for activation of the titin kinase domain during myofibrillogenesis.

The giant muscle protein titin (connectin) is essential in the temporal and spatial control of the assembly of the highly ordered sarcomeres (contractile units) of striated muscle. Here we present the crystal structure of titin's only catalytic domain, an autoregulated serine kinase (titin kinase). The structure shows how the active site is inhibited by a tyrosine of the kinase domain. We describe a dual mechanism of activation of titin kinase that consists of phosphorylation of this tyrosine and binding of calcium/calmodulin to the regulatory tail. The serine kinase domain of titin is the first known non-arginine-aspartate kinase to be activated by phosphorylation. The phosphorylated tyrosine is not located in the activation segment, as in other kinases, but in the P + 1 loop, indicating that this tyrosine is a binding partner of the titin kinase substrate. Titin kinase phosphorylates the muscle protein telethonin in early differentiating myocytes, indicating that this kinase may act in myofibrillogenesis.

Activation of Hypoxia-inducible Factor-1; Definition of Regulatory Domains within the α Subunit

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1alpha and -1beta), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1alpha but not HIF-1beta conferred oxygen-regulated activity. Two minimal domains within HIF-1alpha (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.

Arachidonic Acid Mediates Interferon-γ–Induced Sphingomyelin Hydrolysis and Monocytic Marker Expression in HL-60 Cell Line

AbstractThe biochemical signaling mechanisms involved in transducing the effects of interferon-γ (IFN-γ) on human leukemia-derived HL-60 cell differentiation are not completely understood. Recent studies established the existence of a sphyngomyelin (SM) cycle that operates in response to the action of IFN-γ on HL-60 cells, but the mechanisms by which IFN-γ induces the SM hydrolysis remain unexplored. In this study, biochemical events mediating IFN-γ effects on SM turnover and their specificity and role in HL-60 differentiation were investigated. The activation of the SM cycle by IFN-γ occurred rapidly, with a decrease of approximately 20% in the SM level observed after 60 minutes with a concomitant increase in ceramide level. Treatment of HL-60 cells with IFN-γ did not influence the 1,2-diacylglycerol concentration, intracellular Ca2+ concentration, or phospholipase D activity. IFN-γ stimulated a rapid release of arachidonic acid (AA) from HL-60 cells; the effect was abolished by the pretreatment of cells with pertussis toxin, suggesting a role for a pertussis-toxin–sensitive G protein in IFN-γ–mediated activation of phospholipase A2 (PLA2 ). At 4 to 120 hours after the stimulation of the cells with IFN-γ, a significant increase in the particulate and soluble PLA2 activity was observed, corresponding to an increase in the level of immunoreactive cPLA2 in both cytosol and membrane fractions. The treatment of cells with tyrosine kinase inhibitor herbimycin A completely abolished the effect of IFN-γ on PLA2 activity in membrane and cytosolic fractions, but had no effect on IFN-γ–mediated early AA release suggesting dual mechanism of PLA2 activation. Melittin, potent activator of PLA2 , and AA mimicked the effect of IFN-γ on SM hydrolysis. Pretreatment of HL-60 cells with the PLA2 inhibitor, bromophenacyl bromide (BPB), or pertussis toxin abolished the effect of IFN-γ on SM hydrolysis; exogenous addition of AA overcame the effects of BPB and pertussis toxin. Long-term exposure (5 days) of HL-60 cells to IFN-γ caused an increase in nitroblue tetrazolium (NBT)-reducing and nonspecific esterase (NSE) activity and induced expression of FcγRI (CD64) without significant effects on cell number, adherence, or fagocytic activity. The treatment of cells with AA or melittin induced NBT, NSE, and CD64 expression to the level similar to that observed with IFN-γ, and no further increase was observed with the combination of IFN-γ and AA or IFN-γ and melittin. Treatment of HL-60 cells with indomethacin, an inhibitor of cyclo-oxygenase, and nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, had no effects on IFN-γ–mediated induction of CD64 expression. These studies indicate a key role for the phospholipase A2/AA pathway, as an early biochemical signal elicited by the occupation of IFN-γ–receptor, in mediating IFN-γ induction of the SM cycle and phenotypic changes associated with differentiation of HL-60 along monocytic lineage.

Arachidonic Acid Mediates Interferon-γ–Induced Sphingomyelin Hydrolysis and Monocytic Marker Expression in HL-60 Cell Line

The biochemical signaling mechanisms involved in transducing the effects of interferon-γ (IFN-γ) on human leukemia-derived HL-60 cell differentiation are not completely understood. Recent studies established the existence of a sphyngomyelin (SM) cycle that operates in response to the action of IFN-γ on HL-60 cells, but the mechanisms by which IFN-γ induces the SM hydrolysis remain unexplored. In this study, biochemical events mediating IFN-γ effects on SM turnover and their specificity and role in HL-60 differentiation were investigated. The activation of the SM cycle by IFN-γ occurred rapidly, with a decrease of approximately 20% in the SM level observed after 60 minutes with a concomitant increase in ceramide level. Treatment of HL-60 cells with IFN-γ did not influence the 1,2-diacylglycerol concentration, intracellular Ca2+ concentration, or phospholipase D activity. IFN-γ stimulated a rapid release of arachidonic acid (AA) from HL-60 cells; the effect was abolished by the pretreatment of cells with pertussis toxin, suggesting a role for a pertussis-toxin–sensitive G protein in IFN-γ–mediated activation of phospholipase A2 (PLA2 ). At 4 to 120 hours after the stimulation of the cells with IFN-γ, a significant increase in the particulate and soluble PLA2 activity was observed, corresponding to an increase in the level of immunoreactive cPLA2 in both cytosol and membrane fractions. The treatment of cells with tyrosine kinase inhibitor herbimycin A completely abolished the effect of IFN-γ on PLA2 activity in membrane and cytosolic fractions, but had no effect on IFN-γ–mediated early AA release suggesting dual mechanism of PLA2 activation. Melittin, potent activator of PLA2 , and AA mimicked the effect of IFN-γ on SM hydrolysis. Pretreatment of HL-60 cells with the PLA2 inhibitor, bromophenacyl bromide (BPB), or pertussis toxin abolished the effect of IFN-γ on SM hydrolysis; exogenous addition of AA overcame the effects of BPB and pertussis toxin. Long-term exposure (5 days) of HL-60 cells to IFN-γ caused an increase in nitroblue tetrazolium (NBT)-reducing and nonspecific esterase (NSE) activity and induced expression of FcγRI (CD64) without significant effects on cell number, adherence, or fagocytic activity. The treatment of cells with AA or melittin induced NBT, NSE, and CD64 expression to the level similar to that observed with IFN-γ, and no further increase was observed with the combination of IFN-γ and AA or IFN-γ and melittin. Treatment of HL-60 cells with indomethacin, an inhibitor of cyclo-oxygenase, and nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, had no effects on IFN-γ–mediated induction of CD64 expression. These studies indicate a key role for the phospholipase A2/AA pathway, as an early biochemical signal elicited by the occupation of IFN-γ–receptor, in mediating IFN-γ induction of the SM cycle and phenotypic changes associated with differentiation of HL-60 along monocytic lineage.

Activation of MAP kinase by the LHRH receptor through a dual mechanism involving protein kinase C and a pertussis toxin-sensitive G protein

The LHRH receptor in αT3-1 gonadotrope cells was shown to bring about a marked and sustained activation of MAP kinase. This response was prevented by protein kinase C inhibition or down-regulation and could be partially mimicked by phorbol ester. Additional evidence for inhibition of this response by pertussis toxin and partial mimicry by mastoparan (in a pertussis toxin-sensitive manner) provides the first evidence for G i G o -mediated signal transduction by the LHRH receptor. This dual mechanism of MAP kinase activation appears to be exceptional amongst the G protein-linked receptors that have been investigated.

L-Tryptophan's effects on brain chemistry and sleep in cats and rats: A review

In this review I shall discuss published and unpublished work from my laboratory dealing with L-tryptophan's effects on brain monoamines and sleep in cats and rats. From our work it appears that normal animals may not be suitable subjects for testing sleep-inducing effect of tryptophan since their slow-wave sleep (SWS) latency is relatively short. In polyphasic sleepers like cats, we did not observe tryptophan's hypnotic effect with any dosage used (10, 30 or 135 mg/kg). However, we found small, but statistically significant, sleep-inducing effect of tryptophan (30 mg/kg, IP) in normal rats. We have tried, therefore, to create insomniac cats with long sleep latencies by using methysergide, a serotonin receptor blocker. The results show that in insomniac cats hypnotic effect of tryptophan, a precursor to brain serotonin, was observed. It involved not only reduction of sleep latencies but also an increase in SWS. It seems likely that tryptophan's partial reversal of methysergide's effect in cats occurred via a dual mechanism of serotonergic activation and catecholaminergic deactivation, while its sleep-inducing effect in normal rats may have been due to the attenuation of the activity of brain catecholamines.

Evidence for a dual mechanism of lipolysis activation by epinephrine in rat adipose tissue.

Whole homogenates prepared from tissue previously exposed to epinephrine displayed a 3-fold increased rate of lipolysis of endogenous substrate. When the aqueous infranatant phase of such homogenates was collected by centrifugation and assayed against exogenous triolein emulsions, no hormone effect could be demonstrated. Treatment of such infranatants with cAMP-dependent protein kinase prepared from muscle increased their lipase activity against exogenous triolein by 80%. Employing [3H]triolein emulsions as exogenous substrate, rates of lipolysis of both endogenous and exogenous glycerides were measured simultaneously in whole tissue homogenates. Prior treatment of the tissue with epinephrine increased the rate of lipolysis of endogenous glycerides an average of 3-fold but had no effect on the hydrolysis of exogenous triolein. By contrast, treatment of whole homogenates with protein kinase accelerated lipolysis of exogenous triolein without altering the rate of hydrolysis of endogenous glycerides. The data suggest that a second pathway of lipolysis activation occurs in response to epinephrine in addition to that involving a cAMP-mediated increase in the state of phosphorylation of the hormone-sensitive lipase.