Exercise,free radicals,and lipid peroxidation in type 1 diabetes mellitus

Indirect biochemical techniques have solely been used to ascertain whether type 1 diabetes mellitus patients are more susceptible to resting and exercise-induced oxidative stress. To date there is no direct evidence to support the contention that type 1 diabetic patients have increased levels of free radical species. Thus, the aim of this study was to use electron spin resonance (ESR) spectroscopy in conjunction with alpha-phenyl-tert-butylnitrone (PBN) spin trapping to measure pre- and postexercise free radical concentration in the venous blood of young male patients with type 1 diabetes mellitus (HbA(1c) = 8.2 +/- 1%, n = 12) and healthy matched controls (HbA(1c) = 5.5 +/- 0.2%, n = 13). Supporting measures of lipid peroxidation (malondialdehyde and lipid hydroperoxides), ambient blood glucose and selected antioxidants were also measured. The diabetic patients presented with a comparatively greater concentration of free radicals as measured by ESR and lipid hydroperoxides (LH) compared to the healthy group (p <.05, pooled rest and exercise data), although there was no difference in malondialdehyde (MDA) concentration. alpha-Tocopherol was comparatively lower in the healthy group (p <.05, pooled rest and exercise data vs. diabetic group) due to a selective decrease during physical exercise (p <.05 vs. rest). The hyperfine coupling constants recorded from the ESR spectra (a(Nitrogen) = 1.37 mT and abeta(Hydrogen) = 0.17 mT) are suggestive of either oxygen or carbon-centered species and are consistent with literature values. We suggest that the greater concentration of oxidants seen in the diabetic group may be due to increased glucose autoxidation as a function of this pathology and/or a lower exercise-induced oxidation rate of the major lipid soluble antioxidant alpha-tocopherol. We suggest that the ESR-detected radicals are secondary species derived from decomposition of LH because these are the major initial reaction products of free radical attack on cell membranes.     

Activation of peroxisome proliferator-activated receptor gamma inhibits osteoprotegerin gene expression in human aortic smooth muscle cells

Increasing evidence indicates an important role of PPAR gamma activation in modulating the development and progression of atherosclerosis, however, the mechanisms involved in these effects are not well understood since the PPAR gamma-regulated genes in vascular smooth muscle cells (VSMC) are poorly defined. Here we reported that PPAR gamma ligands, GW7845, ciglitazone and troglitazone had the effect of inhibiting osteoprotegerin (OPG) expression in human aortic smooth muscle cells (HASMC). The effect of GW7845 and ciglitazone on OPG expression was completely abolished by GW9662, a PPAR gamma antagonist. Overexpression of PPAR gamma in HASMC by the infection of a PPAR gamma adenovirus dramatically decreased OPG expression. In addition, PPAR gamma activation inhibited OPG promoter activity. Taken together, our data suggest that OPG expression is a novel PPAR gamma target gene in VSMC and downregulation of OPG expression by PPAR gamma activation provides a new insight into the understanding of the role of PPAR gamma in atheroscelrosis and hypertension.     

IFN-gamma inhibition of TRAIL-induced IAP-2 upregulation, a possible mechanism of IFN-gamma-enhanced TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of TNF family and a potent inducer of apoptosis. The anticancer activities of TNF family members are often modulated by interferon (IFN)-gamma. Thus, we investigated whether IFN-gamma enhances TRAIL-induced apoptosis. We exposed HeLa cells to IFN-gamma for 12 h and then treated with recombinant TRAIL protein. No apoptosis was induced in cells pretreated with IFN-gamma, and TRAIL induced 25% cell death after 3 h treatment. In HeLa cells pretreated with IFN-gamma, TRAIL induced cell death to more than 70% at 3 h, indicating that IFN-gamma pretreatment sensitized HeLa cells to TRAIL-induced apoptosis. We investigated molecules that might be regulated by IFN-gamma pretreatment that would affect TRAIL-induced apoptosis. Western blotting analyses demonstrated that TRAIL treatment increased the level of IAP-2 protein and IFN-gamma pretreatment inhibited the upregulation of IAP-2 protein by TRAIL protein. Our data indicate that TRAIL can signal to activate both apoptosis induction and antiapoptotic mechanism, at least, through IAP-2 simultaneously. IFN-gamma or TRAIL treatment alone did not change expression of other pro- or antiapoptotic proteins such as DR4, DR5, FADD, Bax, IAP-1, XIAP, Bcl-2, and Bcl-XL. Our findings suggest that IFN-gamma may sensitize HeLa cells to TRAIL-induced apoptosis by preventing TRAIL-induced IAP-2 upregulation, and IFN-gamma may play a role in anticancer therapy of TRAIL protein through such mechanism.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Injection of soluble vascular endothelial growth factor receptor 1 into the preovulatory follicle disrupts ovulation and subsequent luteal function in rhesus monkeys

Remarkable changes in vascular permeability and neovascularization occur within the ovulatory, luteinizing follicle. To evaluate the importance of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in periovulatory events, sequential experiments were designed in which vehicle (PBS/0.1% BSA; controls, n = 13) or a low dose (1.5 micro g; n = 4) or a high dose (7.5 micro g; n = 4) of a VEGF antagonist, soluble VEGF receptor 1 (sVEGFR1) chimera, was injected directly into the preovulatory follicle of rhesus monkeys the day before (Day -1) or the day of (Day 0) the midcycle LH surge during spontaneous menstrual cycles. After vehicle injection, animals typically exhibited patterns and levels of serum progesterone (P(4)) that were comparable to those of untreated animals in our colony. Following low-dose sVEGFR1 injection, serum P(4) levels were diminished in two of four animals from the early to midluteal phase, but were similar to vehicle controls thereafter. In contrast, high-dose sVEGFR1 injection decreased serum P(4) levels throughout the luteal phase compared with levels in controls (P < 0.05), but it did not cause premature menstruation. Control follicles displayed indices of rupture (protruding stigmata) and luteinization. However, sVEGFR1-injected follicles exhibited signs of distension (torn surface epithelium/tunica albuginea) and luteinization, but not necessarily timely ovulation. Histological evaluation of serial sections from ovaries removed on Day 3 after treatment revealed that all (n = 3) vehicle-injected follicles ovulated, whereas half (n = 3 of 6) the sVEGFR1-injected follicles failed to ovulate and still contained an oocyte in the antrum. No appreciable differences were apparent between treatment groups in numbers of cells in luteal tissue (Day 3 or 6 after treatment) that stained positive for immunochemical or histochemical markers of proliferative (Ki67), endothelial (platelet endothelial cell adhesion molecule 1), and steroidogenic (3beta-hydroxysteroid dehydrogenase) cells. However, there was a dose-dependent increase (P < 0.05) in extracellular space in the corpus luteum by midluteal phase in sVEGFR1-treated animals. The data suggest that acute exposure to a VEGF antagonist can impair ovulation, and the subsequent development and functional capacity of the primate corpus luteum. The results are consistent with a critical role for VEGF in normal ovarian function during the periovulatory interval in primates.     

The brownian ratchet and power stroke models for posttranslational protein translocation into the endoplasmic reticulum

A quantitative analysis of experimental data for posttranslational translocation into the endoplasmic reticulum is performed. This analysis reveals that translocation involves a single rate-limiting step, which is postulated to be the release of the signal sequence from the translocation channel. Next, the Brownian ratchet and power stroke models of translocation are compared against the data. The data sets are simultaneously fit using a least-squares criterion, and both models are found to accurately reproduce the experimental results. A likelihood-ratio test reveals that the optimal fit of the Brownian ratchet model, which contains one fewer free parameter, does not differ significantly from that of the power stroke model. Therefore, the data considered here cannot be used to reject this import mechanism. The models are further analyzed using the estimated parameters to make experimentally testable predictions.     

Noncontact dipole effects on channel permeation. VI. 5F- and 6F-Trp gramicidin channel currents

Fluorination of peptide side chains has been shown to perturb gramicidin channel conductance without significantly changing the average side chain structure, which, it is hoped, will allow detailed analysis of electrostatic modulation of current flow. Here we report a 1312-point potassium current-voltage-concentration data set for homodimeric channels formed from gramicidin A (gA) or any of eight fluorinated Trp analogs in both lecithin and monoglyceride bilayers. We fit the data with a three-barrier, two-site, two-ion (3B2S) kinetic model. The fluorination-induced changes in the rate constants were constrained by the same factor in both lipids. The rate constant changes were converted to transition-state free-energy differences for comparison with previous electrostatic potential energy differences based on an ab initio force field. The model allowed a reasonably good fit (chi = 8.29 with 1271 degrees of freedom). The measured changes were subtle. Nevertheless, the fitted energy perturbations agree well with electrostatic predictions for five of the eight peptides. For the other three analogs, the fitted changes suggested a reduced translocation barrier rather than the reduced exit barrier as predicted by electrostatics.     

Uniformity,ideality, and hydrogen bonds in transmembrane alpha-helices

Protein environments substantially influence the balance of molecular interactions that generate structural stability. Transmembrane helices exist in the relatively uniform low dielectric interstices of the lipid bilayer, largely devoid of water and with a very hydrophobic distribution of amino acid residues. Here, through an analysis of bacteriorhodopsin crystal structures and the transmembrane helix structure from M2 protein of influenza A, some helices are shown to be exceptionally uniform in hydrogen bond geometry, peptide plane tilt angle, and backbone torsion angles. Evidence from both the x-ray crystal structures and solid-state NMR structure suggests that the intramolecular backbone hydrogen bonds are shorter than their counterparts in water-soluble proteins. Moreover, the geometry is consistent with a dominance of electrostatic versus covalent contributions to these bonds. A comparison of structure as a function of resolution shows that as the structures become better characterized the helices become much more uniform, suggesting that there is a possibility that many more uniform helices will be observed, even among the moderate resolution membrane protein structures that are currently in the Protein Data Bank that do not show such features.     

Solid-state 19F-NMR analysis of 19F-labeled tryptophan in gramicidin A in oriented membranes

The response of membrane-associated peptides toward the lipid environment or other binding partners can be monitored by solid-state NMR of suitably labeled side chains. Tryptophan is a prominent amino acid in transmembrane helices, and its (19)F-labeled analogues are generally biocompatible and cause little structural perturbation. Hence, we use 5F-Trp as a highly sensitive NMR probe to monitor the conformation and dynamics of the indole ring. To establish this (19)F-NMR strategy, gramicidin A was labeled with 5F-Trp in position 13 or 15, whose chi(1)/chi(2) torsion angles are known from previous (2)H-NMR studies. First, the alignment of the (19)F chemical shift anisotropy tensor within the membrane was deduced by lineshape analysis of oriented samples. Next, the three principal axes of the (19)F chemical shift anisotropy tensor were assigned within the molecular frame of the indole ring. Finally, determination of chi(1)/chi(2) for 5F-Trp in the lipid gel phase showed that the side chain alignment differs by up to 20 degrees from its known conformation in the liquid crystalline state. The sensitivity gain of (19)F-NMR and the reduction in the amount of material was at least 10-fold compared with previous (2)H-NMR studies on the same system and 100-fold compared with (15)N-NMR.     

Studies of the receptor-bound conformation of alphaIIbbeta3 antagonists by 15N-edited NMR spectroscopy

We report the results of NMR studies and computer simulations of potent antagonists reflective of the alpha(IIb)beta(3) receptor-bound conformations. The peptides c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Phe(4)-(15)N-Arg(5)-Cys]-NH(2) (Phe-Arg analog) (Mpa: 3-mercaptopropionic acid) and c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Asp(4)-(15)N-Val(5)-Cys]-NH(2) (Asp-Val analog) were subjected to (15)N-edited NMR experiments to study the conformations of these peptides in the absence and in the presence of alpha(IIb)beta(3) receptor. The NMR studies of the Phe-Arg analog, a selective alpha(IIb)beta(3) antagonist, resulted in distinctly different experimental data in the presence and absence of the receptor. The computer simulations for this peptide resulted in one large family of structures consistent with the experimental data. This conformation suggests a type I beta-turn spanning residues Arg(1) and Gly(2) when bound to the receptor and we were able to establish a model for the three dimensional arrangement of the pharmacophores. The studies on the Asp-Val analog, an alpha(v)beta(3) antagonist that binds to the alpha(IIb)beta(3) with moderate affinity, resulted in conformations that are not as well defined as those for the Phe-Arg analog but are consistent with the model established for this analog. These results are important for the design of novel alpha(IIb)beta(3) antagonists.