Kinetics and activation parameter analyses of hydrolysis and interconversion of 2',5'- and 3',5'-linked dinucleoside monophosphate at extremely high temperatures

Kinetic analysis of hydrolytic stability of 2',5'- and 3',5'-linked dinucleoside monophosphate (N(2)'pN and N(3)'pN) was successfully performed in aqueous solution at 175-240 degrees C using a new real-time monitoring method for rapid hydrothermal reactions. The half-lives of NpN were in the range 2-8 s at 240 degrees C and apparent activation energy decreases in the order U(2)'pU>A(2)'pA>G(2)'pG>U(3)pU approximately C(3)'pC>A(3)pA. The stability of phosphodiester bond was dependent on the types of base moiety and phosphodiester linkages, but no systematic correlation was found between the structure and stability. The interconversion of 2',5'-adenylyladenosine monophosphate (A(2)'pA) and 3',5'-adenylyladenosine monophosphate (A(3)'pA) was enhanced in the presence of D- or L-histidine. The rate constants of degradation of NpN were dissected into the rate constants of hydrolysis and interconversion between N(2)'pN and N(3)'pN using a computer program SIMFIT. Kinetic analysis supports the mechanism that imidazolium ion and imidazole catalyze interconversion and hydrolysis even under hydrothermal environments. The activation parameters for the hydrolysis and interconversion of NpN were systematically determined for the first time from the temperature dependence of the rate constants, where both DeltaH(app)( not equal ) and DeltaS(app)( not equal ) for 2',5'-linked NpN are larger than those for 3',5'-linked NpN. These parameters support the pseudorotation mechanism through pentacoordinate intermediate from 2',5'- and 3',5'-linked NpN, where the average value of DeltaH( not equal ) (pseudorotation) was estimated to be 30+/-18 kJ mol(-1) at 175-240 degrees C.     

Spermine induces cataract and 43-kDa protein that binds spermine possibly participates in the cataract formation

Among polyamines (putrescine, spermidine, and spermine), spermine specifically induces cataract in an organ cultured lens. Spermine uptake nearly paralleled the cataract formation. When polyamines were added to lens soluble proteins, spermine specifically induced turbidity. When lens soluble proteins were separated by gel chromatography, heavy-molecular-weight protein (HMW, high molecular form of alpha-crystallin) and proteins between betaH- and betaL-crystallin fractions reacted with spermine and aggregated. SDS-polyacrylamide gel electrophoresis of the aggregated proteins showed that 43-kDa lens protein was commonly observed in both aggregates. Spermine-affinity chromatography of the total soluble proteins showed the binding of HMW protein to the gel and the chromatogram of the second turbidity peak in the gel chromatography showed the binding of 43-kDa protein. These results indicated that 43-kDa protein, which is present as a subunit in HMW and also in free form, binds spermine and induces turbidity of lens soluble proteins and produces cataract in a cultured lens.     

Formation of superoxide anion during ferrous ion-induced decomposition of linoleic acid hydroperoxide under aerobic conditions

We studied the mechanism of formation of oxygen radicals during ferrous ion-induced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2*-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2*-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O*)R2] is converted into the hydroxylcarbon radical [R1C*(OH)R2] in water, and that this carbon radical reacts with oxygen to give the alpha-hydroxyperoxyl radical [R1R2C(OH)OO*], which decomposes into the carbocation [R1C+(OH)R2] and O2*-.     

Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.     

Melatonin reduces cerebral edema formation caused by transient forebrain ischemia in rats

Reduction of cerebral edema, an early symptom of ischemia, is one of the most important remedies for reducing subsequent chronic neural damage in stroke. Melatonin, a metabolite of tryptophan released from the pineal gland, has been found to be effective against neurotoxicity in vitro. The present study was aimed to demonstrate the effectiveness of melatonin in vivo in reducing ischemia-induced edema using magnetic resonance imaging (MRI). Rats were subjected to middle cerebral artery (MCA) occlusion/reperfusion surgery. Melatonin was administered twice (6.0 mg/kg, p.o.): just prior to 1 h MCA occlusion and 1 day after the surgery. T2-weighted multislice spin-echo images were acquired 1 day after the surgery. Increases in T2-weighted signals in ischemic sites of the brain were clearly observed after MCA occlusion. The signal increase was found mainly in the striatum and in the cerebral cortex in saline-treated control rats. In the melatonin-treated group, the total volume of cerebral edema was reduced by 45.3% compared to control group (P < 0.01). The protective effect of melatonin against cerebral edema was more clearly observed in the cerebral cortex (reduced by 56.1%, P < 0.01), while the reduction of edema volume in the striatum was weak (reduced by 23.0%). The present MRI study clearly demonstrated that melatonin is effective in reducing edema formation in ischemic animals in vivo, especially in the cerebral cortex. Melatonin may be highly useful in preventing cortical dysfunctions such as motor, sensory, memory, and psychological impairments.     

Paraquat- and diquat-induced oxygen radical generation and lipid peroxidation in rat brain microsomes

NADPH-menadione reductase activity by rat brain microsomes (Ms) was decreased 40-50% by 10 microM dicumarol, a potent inhibitor of DT-diaphorase, whereas no change in NADPH-paraquat (PQ) and -diquat (DQ) reductase activity was observed. NADPH-DQ reductase activity in brain Ms was 2.5-fold higher than NADPH-PQ reductase activity. The formation of PQ and DQ radicals was verified optically and observed directly by ESR spectroscopy in the NADPH-PQ and -DQ reductase reactions by brain Ms under anaerobic conditions. PQ- and DQ-induced superoxide formation was confirmed by the detection of DMPO-OOH ESR signals and followed by chemiluminescence (CL) of a Cypridina luciferin analogue (CLA). The kinetics and intensity of the CL were consistent with the observations that the reduction in DQ is faster than that in PQ. Thiobarbituric acid reactive substances (TBARS) and phospholipid hydroperoxides in brain Ms increased in the presence of NADPH and Fe3+. The generation of both lipid peroxidation products derived from brain Ms decreased with increasing concentrations of PQ and DQ. The inhibitory effect of DQ is more pronounced than that of PQ. The formation of PQ- and DQ-induced reactive oxygen species was not associated with lipid peroxidation in rat brain Ms.     

Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression

Fractalkine/CX3C ligand 1 and its receptor CX3CR1 are known to mediate both cell adhesion and cell migration. Here we show that CX3CR1 defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B, which include NK cells, gammadelta T cells, and terminally differentiated CD8(+) T cells. In addition, soluble fractalkine preferentially induced migration of cytotoxic effector lymphocytes. Furthermore, interaction of cytotoxic effector lymphocytes with membrane-bound fractalkine promoted subsequent migration to the secondary chemokines, such as macrophage inflammatory protein-1beta/CC ligand 4 or IL-8/CXC ligand 8. Thus, fractalkine expressed on inflamed endothelium may function as a vascular regulator for cytotoxic effector lymphocytes, regardless of their lineage and mode of target cell recognition, through its ability to capture them from blood flow and to promote their emigration in response to other chemokines.     

Reversal by dithiothreitol treatment of the block in murine leukemia virus maturation induced by disulfide cross-linking

We previously reported that if murine leukemia virus particles are produced in the presence of the mild oxidizing agent disulfide-substituted benzamide-2, they fail to undergo the normal process of virus maturation. We now show that treatment of these immature particles with a reducing agent (dithiothreitol) induces their maturation in vitro, as evidenced by proteolytic cleavage of Gag, Gag-Pol, and Env proteins and by their morphology. The identification of partial cleavage products in these particles suggests the sequence with which the cleavages occur under these conditions. This may be a useful experimental system for further analysis of retroviral maturation under controlled conditions in vitro.