The 21-aminosteroid U74389G prevents the down-regulation and decrease in activity of CYP1A1, 1A2 and 3A6 induced by an inflammatory reaction

In vivo, the 21-aminosteroid U74389G prevents the decrease in cytochrome P450 (P450) activity produced by a turpentine-induced inflammatory reaction (TIIR). To investigate the underlying mechanism of action, four groups of rabbits were used, controls receiving or not U74389G, and rabbits with the inflammatory reaction receiving or not U74389G. Hepatocytes were isolated 48h later and incubated for 4 and 24h with the serum of the rabbits. In vivo, the TIIR diminished CYP1A1/2 and 3A6 expression, and enhanced hepatic malondialdehyde (MDA) and nitric oxide (NO*) concentrations (p<0.05). U74389G prevented the increase in MDA, as well as the decrease in CYP1A1/2 amounts and activity, but increased CYP3A6 expression by 40% (p<0.05). In vitro, compared with serum from control rabbits (S(CONT)), incubation of serum from rabbits with TIIR (S(TIIR)) for 4 and 24h with hepatocytes from rabbits with TIIR (H(TIIR)) reduced CYP1A2 and CYP3A6 activity (p<0.05) and increased the formation of NO* and MDA. In rabbits with TIIR pretreated with U74389G, the S(TIIR+U) failed to reduce CYP1A2 activity or to increase MDA, although increased NO* and further reduced CYP3A6 activity. On the other hand, in hepatocytes harvested from rabbits with TIIR pretreated with U74389G, S(TIIR) did not decrease CYP1A2 activity and did not enhance MDA, but still increased NO*. In vitro, the reduction of CYP1A2 and CYP3A6 activity by S(TIIR) is not associated to NF-kappaB activation. In conclusion, U74389G prevents CYP1A1/2 down-regulation and decrease in activity by a double mechanism: hindering the release of serum mediators and by averting intracellular events, effect possibly associated with its antioxidant activity. On the other hand, U74389G up-regulates CYP3A6 but inhibits its catalytic activity.     

Regulation of lipid raft proteins by glimepiride- and insulin-induced glycosylphosphatidylinositol-specific phospholipase C in rat adipocytes

The insulin receptor-independent insulin-mimetic signalling provoked by the antidiabetic sulfonylurea drug, glimepiride, is accompanied by the redistribution and concomitant activation of lipid raft-associated signalling components, such as the acylated tyrosine kinase, pp59(Lyn), and some glycosylphosphatidylinositol-anchored proteins (GPI-proteins). We now found that impairment of glimepiride-induced lipolytic cleavage of GPI-proteins in rat adipocytes by the novel inhibitor of glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), GPI-2350, caused almost complete blockade of (i) dissociation from caveolin-1 of pp59(Lyn) and GPI-proteins, (ii) their redistribution from high cholesterol- (hcDIGs) to low cholesterol-containing (lcDIGs) lipid rafts, (iii) tyrosine phosphorylation of pp59(Lyn) and insulin receptor substrate-1 protein (IRS-1) and (iv) stimulation of glucose transport as well as (v) inhibition of isoproterenol-induced lipolysis in response to glimepiride. In contrast, blockade of the moderate insulin activation of the GPI-PLC and of lipid raft protein redistribution by GPI-2350 slightly reduced insulin signalling and metabolic action, only. Importantly, in response to both insulin and glimepiride, lipolytically cleaved hydrophilic GPI-proteins remain associated with hcDIGs rather than redistribute to lcDIGs as do their uncleaved amphiphilic versions. In conclusion, GPI-PLC controls the localization within lipid rafts and thereby the activity of certain GPI-anchored and acylated signalling proteins. Its stimulation is required and may even be sufficient for insulin-mimetic cross-talking to IRS-1 in response to glimepiride via redistributed and activated pp59(Lyn).     

Cyclooxygenase-2 Inhibition Delays the Attainment of Peak Woven Bone Formation following Four-Point Bending in the Rat

Fracture healing is retarded in the presence of cyclooxygenase-2 (COX-2) inhibitors, demonstrating an important role of COX-2 in trauma-induced woven bone adaptation. The aim of this experiment was to determine the influence of COX-2 inhibition on the remodeling and consolidation of nontraumatic woven bone produced by mechanical loading. A periosteal woven bone callus was initiated in the right tibia of female Wistar rats following a single bout of four-point bending, applied as a haversine wave for 300 cycles at a frequency of 2 Hz and a magnitude of 65 N. Daily injections of vehicle (VEH, polyethylene glycol) or the COX-2 inhibitor 5,5-dimethyl-3-3(3 fluorophenyl)-4-(4-methylsulfonal)phenyl-2(5H)-furanone (DFU, 2.0 mg · kg⁻¹ and 0.02 mg · kg⁻¹ i.p.), commenced 7 days postloading, and tibiae were examined 2, 3, 4, and 5 weeks postloading. Tibiae were dissected, embedded in polymethylmethacrylate, and sectioned for histomorphometric analysis of periosteal woven bone. No significant difference in peak woven bone area was observed between DFU-treated and VEH rats. However, treatment with DFU resulted in a temporal defect in woven bone formation, where the achievement of peak woven bone area was delayed by 1 week. Woven bone remodeling was observed in DFU-treated rats at 21 days postloading, demonstrating that remodeling of the periosteal callus is not prevented in the presence of a COX-2 inhibitor in the rat. We conclude that COX-2 inhibition does not significantly disrupt the mechanism of woven bone remodeling but alters its timing.     

Effect of beta-dystroglycan processing on utrophin/Dp116 anchorage in normal and mdx mouse Schwann cell membrane

In the peripheral nervous system, utrophin and the short dystrophin isoform (Dp116) are co-localized at the outermost layer of the myelin sheath of nerve fibers; together with the dystroglycan complex. Dp116 is associated with multiple glycoproteins, i.e. sarcoglycans, and alpha- and beta-dystroglycan, which anchor the cytoplasmic protein subcomplex to the extracellular basal lamina. In peripheral nerve, matrix metalloproteinase activity disrupts the dystroglycan complex by cleaving the extracellular domain of beta-dystroglycan. Metalloproteinase creates a 30 kDa fragment of beta-dystroglycan, leading to a disruption of the link between the extracellular matrix and the cell membrane. Here we asked if the processing of the beta-dystroglycan could influence the anchorage of Dp116 and/or utrophin in normal and mdx Schwann cell membrane. We showed that metalloproteinase-9 was more activated in mdx nerve than in wild-type ones. This activation leads to an accumulation of the 30 kDa beta-dystroglycan isoform and has an impact on the anchorage of Dp116 and utrophin isoforms in mdx Schwann cells membrane. Our results showed that Dp116 had greater affinity to the full length form of beta-dystroglycan than the 30 kDa form. Moreover, we showed for the first time that the short isoform of utrophin (Up71) was over-expressed in mdx Schwann cells compared with wild-type. In addition, this utrophin isoform (Up71) seems to have greater affinity to the 30 kDa beta-dystroglycan which could explain the increased stabilization of this 30 kDa form at the membrane compartment. Our results highlight the potential participation of the short utrophin isoform and the cleaved form of beta-dystroglycan in mdx Schwann cell membrane architecture. We proposed that these two proteins could be implicated in Schwann cell proliferation in response to a microenvironment stress such as mediated by accumulating macrophages in mdx mouse muscle inflammation sites.     

The Na,K-ATPase alpha4 isoform from humans has distinct enzymatic properties and is important for sperm motility

In the rat, the Na,K-ATPase alpha4 isoform exhibits unique enzymatic characteristics and is important for sperm motility. In this work, we studied expression, localization and function of alpha4 in human spermatozoa. We show two catalytically active Na,K-ATPase alpha polypeptides with different ouabain affinity and identified expression of alpha1, alpha4, beta1 and beta3 isoforms in the gametes. In addition, human sperm presented two Na,K-ATPases composed of alpha4, alpha4beta1 and alpha4beta3. Kinetic analysis of these isozymes produced in insect cells showed that, compared with human alpha1beta1, alpha4beta1 and alpha4beta3 exhibit higher Na(+) and lower K(+) affinity and higher sensitivity to ouabain. These particular enzymatic properties suggested a role for alpha4 in sperm function. Using computer-assisted sperm analysis (CASA), we found that ouabain inhibition of alpha4 significantly decreased percentage sperm motility. In contrast, ouabain did not affect linearity of forward progression, amplitude of lateral head displacement, beat cross frequency and sperm straight-line, curvilinear or average path velocities. This suggests a primary role of alpha4 in flagellar motility. Accordingly, we found alpha4 in the sperm tail, predominating in the mid-piece of the flagellum. Therefore, similar to the rat ortholog, human Na,K-ATPase alpha4 isoform has a distinct activity that is essential for sperm function.     

Divergent actions of the pyrethroid insecticides S-bioallethrin, tefluthrin, and deltamethrin on rat Nav1.6 sodium channels

We expressed rat Na_v1.6 sodium channels in combination with the rat β₁ and β₂ auxiliary subunits in Xenopus laevis oocytes and evaluated the effects of the pyrethroid insecticides S-bioallethrin, deltamethrin, and tefluthrin on expressed sodium currents using the two-electrode voltage clamp technique. S-Bioallethrin, a type I structure, produced transient modification evident in the induction of rapidly decaying sodium tail currents, weak resting modification (5.7% modification at 100 μM), and no further enhancement of modification upon repetitive activation by high-frequency trains of depolarizing pulses. By contrast deltamethrin, a type II structure, produced sodium tail currents that were ~ 9-fold more persistent than those caused by S-bioallethrin, barely detectable resting modification (2.5% modification at 100 μM), and 3.7-fold enhancement of modification upon repetitive activation. Tefluthrin, a type I structure with high mammalian toxicity, exhibited properties intermediate between S-bioallethrin and deltamethrin: intermediate tail current decay kinetics, much greater resting modification (14.1% at 100 μM), and 2.8-fold enhancement of resting modification upon repetitive activation. Comparison of concentration-effect data showed that repetitive depolarization increased the potency of tefluthrin ~ 15-fold and that tefluthrin was ~ 10-fold more potent than deltamethrin as a use-dependent modifier of Na_v1.6 sodium channels. Concentration-effect data from parallel experiments with the rat Na_v1.2 sodium channel coexpressed with the rat β₁ and β₂ subunits in oocytes showed that the Na_v1.6 isoform was at least 15-fold more sensitive to tefluthrin and deltamethrin than the Na_v1.2 isoform. These results implicate sodium channels containing the Na_v1.6 isoform as potential targets for the central neurotoxic effects of pyrethroids.     

原肌球蛋白的分子生物学研究进展

原肌球蛋白 (TM )是肌肉收缩过程中重要的调节蛋白质 ,它以大量异构体形式广泛分布于各种真核细胞中。哺乳动物中的 4个TM基因已被确认 ,分别命名为TPM1、TPM2 、TPM3、TPM4 ,至少可表达出 2 0种TM异构体。TPM1基因突变与家族性肥大性心肌病有关 ;TPM3基因突变与线状肌病和骨骼肌无力有关