Time Course Of Protection Research Articles

Trimer-to-Monomer Disruption Mechanism for a Potent, Protease-Resistant Antagonist of Tumor Necrosis Factor-α Signaling.

Aberrant tumor necrosis factor-α (TNFα) signaling is associated with many inflammatory diseases. The homotrimeric quaternary structure of TNFα is essential for receptor recognition and signal transduction. Previously, we described an engineered α/β-peptide inhibitor that potently suppresses TNFα activity and resists proteolysis. Here, we present structural evidence that both the α/β-peptide inhibitor and an all-α analogue bind to a monomeric form of TNFα. Calorimetry data support a 1:1 inhibitor/TNFα stoichiometry in solution. In contrast, previous cocrystal structures involving peptide or small-molecule inhibitors have shown the antagonists engaging a TNFα dimer. The structural data reveal why our inhibitors favor monomeric TNFα. Previous efforts to block TNFα-induced cell death with peptide inhibitors revealed that surfactant additives to the assay conditions cause a more rapid manifestation of inhibitory activity than is observed in the absence of additives. We attributed this effect to a loose surfactant TNFα association that lowers the barrier to trimer dissociation. Here, we used the new structural data to design peptide inhibitors bearing a surfactant-inspired appendage intended to facilitate TNFα trimer dissociation. The appendage modified the time course of protection from cell death.

Olfactory Ensheathing Cells Rescue Optic Nerve Fibers in a Rat Glaucoma Model

To determine if transplantation of olfactory ensheathing cells (OECs) can reduce loss of optic nerve axons after raised intraocular pressure (IOP) in the rat. OECs cultured from the adult olfactory mucosa were transplanted into the region of the optic disc. The IOP was raised by injection of magnetic microspheres into the anterior chamber. At 4 weeks after raising the IOP, the transplanted OECs had migrated into the dorsal area of the optic nerve head (ONH) where they surrounded the optic nerve fibers with a non-myelinated ensheathment. The mean amount of damage to the ONH astrocytic area in rats was 51.0% compared with 85.8% in those without OEC transplants (P < 0.02) and the mean loss of axons in the optic nerve was 51.0% compared with 80.3% in the absence of OECs (P < 0.01). OECs transplanted into the region of the ONH of the rat can reduce the loss of axons and the damage to ONH astrocytes caused by raised IOP. Confirmation of these preliminary experimental data, further understanding of possible mechanisms of axonal protection by OECs, and the longer-term time course of protection could provide a basis for future human clinical trials of autografted OECs, which would be available from autologous nasal epithelial biopsies.

Effects of intrastriatal GDNF on the response of dopamine neurons to 6-hydroxydopamine: Time course of protection and neurorestoration

Glial cell line-derived neurotrophic factor (GDNF) protects dopamine (DA) neurons from 6-hydroxydopamine (6-OHDA) toxicity. We have now explored this protection over 8weeks following toxin administration. Infusion of Fluoro-Gold (FG) into the striatum was followed 1week later by GDNF (9μg) or its vehicle. Six hours later, animals received 6-OHDA (4μg) into the same site. 6-OHDA caused a loss of cells in the substantia nigra that expressed both FG and tyrosine hydroxylase (TH) and striatal terminals expressing TH, the high affinity dopamine transporter (DAT), and the vesicular monoamine transporter 2 (VMAT2) as assessed 2–8weeks later. Loss of FG+ cells, and striatal DA was completely blocked by GDNF by 2weeks. In contrast, GDNF only slightly attenuated the loss of TH, DAT, or VMAT2 in the striatum at 2weeks, but had restored these markers by 4–8weeks. Thus, GDNF prevents DA cell death and loss of striatal DA content, but several weeks are required to fully restore the dopaminergic phenotype. These results provide insight into the mechanism of GDNF protection of DA neurons, and may help avoid incorrect interpretations of temporary phenotypic changes.

Time Course of Neurotrophic Factor Upregulation and Retinal Protection against Light-Induced Damage after Optic Nerve Section

To assess neurotrophic factor upregulation in the retina after damage to the optic nerve and relate that regulation to changes in photoreceptor stability and function. Retinas of adult pigmented (Long-Evans) rats were examined at successive times (1-60 days) after unilateral optic nerve section. The distribution and expression of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (FGF-2) and their receptor elements FGFR1 and CNTFRalpha were studied with immunohistochemistry and Western blot analysis. FGF-2 and CNTF mRNA levels were also assessed, with semiquantitative reverse transcription-PCR. Levels and localization of the intracellular signaling molecule ERK and its activated, phosphorylated form pERK, were examined by immunohistochemistry. To assess the correlation between neurotrophic factor levels and their protective effect against light damage, albino (Sprague-Dawley) rats were exposed to bright continuous light (1000 lux) for 24 or 48 hours at successive times after nerve section. The TUNEL technique was used to visualize neuronal cell death in the retina. CNTF upregulation was detected 1 week after optic nerve section, peaked at 2 weeks, and fell to control levels at 4 weeks. CNTF appeared first in the inner retina in the ganglion cells, then in the Muller cells in which it became prominent at the outer limiting membrane (OLM) and in the outer segment (OS) region of photoreceptors. FGF-2 upregulation became prominent, particularly in photoreceptors, 21 to 28 days after surgery, continued to 2 months, and slowly declined thereafter. Double labeling with antibodies to ligand and the receptor showed colocalization of CNTF to its receptor at the OS region, whereas FGF-2-to-FGFR1 binding was found in the outer nuclear (ONL) and outer plexiform (OPL) layers. Optic nerve section provided a significant protective effect against light-induced damage in the first 2 weeks. There was no protection when animals were exposed to damaging light 1 month after nerve section. The upregulation of CNTF 7 to 14 days after nerve section correlates with a reduction in the a-wave described previously. Colocalization of CNTF and CNTFRalpha on the outer segments suggests that CNTF acts at the photoreceptor membrane. The slower upregulation of FGF-2 correlates with a reduction of the b-wave. FGF-2/FGFR1 colocalization in the OPL suggests that this factor acts at the synaptic terminals of photoreceptors, modulating the release of neurotransmitters. The time course of pERK upregulation suggests that the successive upregulation of CNTF and FGF-2 activates the ERK pathway. Based on the time course of protection against bright continuous light, it seems that CNTF plays a major role in this effect, and FGF-2 has a less important role in the protection against light-induced damage.

Metallothionein Protects Islets from Hypoxia and Extends Islet Graft Survival by Scavenging Most Kinds of Reactive Oxygen Species

Islet transplantation is a promising therapy for Type 1 diabetes, but many attempts have failed due to early graft hypoxia or immune rejection, which generate reactive oxygen species (ROS). In the current study, we determined that transgenic overexpression of the antioxidant metallothionein (MT) in pancreatic beta cells provided broad resistance to oxidative stress by scavenging most kinds of ROS including H2O2, peroxynitrite radical released from streptozotocin, 3-morpholinosydnonimine (SIN-1), and superoxide radical produced by xanthine/xanthine oxidase. MT also reduced nitric oxide-induced beta cell death. A direct test of hypoxia/reperfusion sensitivity was made by exposing FVB and MT islets to hypoxia (1% O2). MT markedly reduced ROS production and improved islet cell survival. Because MT protected beta cells from a broad spectrum of ROS and from hypoxia, we considered it to be an ideal candidate for improving islet transplantation. We first tested syngeneic transplantation by implanting islets under the kidney capsule of the same strain, FVB mice, thereby eliminating the immune rejection component. Under these conditions, MT islets maintained much greater insulin content than control islets. Allotransplantation was then tested. MT transgenic and normal FVB islets were implanted under the kidney capsule of BALB/c mice that were previously treated with streptozotocin to induce diabetes. We found that MT islets extended the duration of euglycemia 2-fold longer than nontransgenic islets. The benefit of MT was due to protection from ROS since nitrotyrosine staining, an indicator of free radical damage, was much lower in MT grafts than in FVB grafts. The time course of protection suggested that the major mode of MT action may have been protection from hypoxia or hypoxia/reperfusion. These data demonstrate that treatment with a broad spectrum antioxidant protects islets from ROS damage such as that produced during the early phase of islet transplantation.

Infarct Size Reduction by Ischemic Preconditioning Is a Monophasic, Short-Lived Phenomenon in Anesthetized Pigs.

BACKGROUND: Controversy exists concerning the duration of infarct size reduction with ischemic preconditioning in different species. In the present study, we (a) evaluated the time course of protection with preconditioning and (b) sought to determine whether late protection (the "second window") after 24 hours is manifest in the open-chest pig model. METHODS AND RESULTS: Six groups of pentobarbital-anesthetized pigs underwent 1 hour of left anterior descending coronary artery occlusion and 2 hours of reperfusion. Group 1 served as control, and pigs in group 2 received two 10-minute episodes of preconditioning ischemia followed by 30 minutes of reperfusion before the sustained 1-hour occlusion. In groups 3-6, the period of intervening reperfusion between the preconditioning stimulus and the index ischemia was extended to 60, 90, and 300 minutes and 24 hours, respectively. The area at risk was determined by fluorescein dye injection, and infarct size was measured by incubation in p-nitrobluetetrazolium and expressed as percent of the risk area. Infarct size in preconditioned pigs (group 2) was significantly reduced compared with controls (25.6 +/- 3.9% v 71.3 +/- 5.9%, P <.001). Extension of the intervening reperfusion to 60, 90, and 300 minutes and 24 hours resulted in infarct sizes of 64.5 +/- 5.5%, 67.2 +/- 8%, 62.6 +/- 6.1%, and 75.3 +/- 7%, respectively (P = NS v control). CONCLUSIONS: The infarct size-limiting effects of ischemic preconditioning last less than 1 hour in the pig model. Moreover, in contrast to other species, a late protection at 24 hours after the preconditioning stimulus was not detected. These results indicate that precondition-induced reduction of infarct size is monophasic in anesthetized pigs.

Preconditioning with a single short episode of global ischemia in the isolated working rat heart: effect on structure, mechanical function, and energy metabolism for various durations of sustained global ischemia.

Preconditioning in the setting of global ischemia, using functional recovery during reperfusion as the endpoint, has recently been demonstrated in the isolated perfused rat heart. It has been suggested that its beneficial actions have a metabolic basis. The isolated rat heart has not been fully characterized with respect to the metabolic, functional, and structural changes associated with this phenomenon in the setting of global ischemia. The purpose of this study was to determine (1) the time course of protection conferred by a single episode (5 minutes) of preconditioning; (2) changes in tissue high energy phosphates, lactate, and glycogen levels at different time intervals; and (3) morphological appearance of the heart at the end of ischemia as well as after reperfusion. Isolated perfused working rat hearts were used. Preconditioning consisted of a single episode of 5 minutes of global ischemia and 15 minutes of reperfusion. Preconditioned and non-preconditioned hearts were subjected to global ischemia of 20-35 minutes duration. Functional recovery, energy metabolism (high energy phosphates, lactate, and glycogen), and structural appearance were studied at different stages. The functional recovery of the preconditioned hearts was significantly higher than in the corresponding nonpreconditioned group during reperfusion for all durations of ischemia longer than 25 minutes. The degree of protection observed was less than reported previously. A minor degree of energy sparing was reflected by differences in the rate of depletion of glycogen and accumulation of tissue lactate during the sustained episode of ischemia. Semiquantitative light microscopy evaluation revealed that ischemia-induced structural damage was less in the preconditioned hearts, both at the end of the sustained ischemic episode as well as after reperfusion. A single episode of global ischemia successfully preconditions the isolated working rat heart. The protection elicited was demonstrated on a functional and structural level, and was accompanied by a small energy-sparing effect.

The protective effect of damaging eccentric exercise against repeated bouts of exercise in the mouse tibialis anterior muscle.

Using a damaging eccentric exercise regime of the mouse tibialis anterior (TA) muscle we have investigated the extent and time course of protection afforded by one bout of exercise against damage resulting from a second bout of activity. Maximal force and fibre morphology were preserved if the exercise was repeated within 21 days, but by 84 days muscles once again became susceptible to damage. Low-frequency force loss had a shorter time course of protection against repeated exercise, lasting less than 21 days. The results provide evidence for different mechanisms contributing to the development of muscle damage following eccentric exercise and provide a basis for characterizing the adaptive response of muscle to damaging exercise.

Early hydrogen-bonding events in the folding reaction of ubiquitin.

The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogen-deuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 back-bone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the beta-sheet and the alpha-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slow-folding species are less prominent.

Differential time course of protection by monoamine oxidase inhibition and uptake inhibition against MPTP neurotoxicity on central catecholamine neurons in mice

Pretreatment with the monoamine oxidase inhibitor pargyline or the catecholamine uptake blocker nomifensine both protected central catecholamine neurons against MPTP-induced neurotoxicity as monitored by analyzing catecholamine levels and [ 3H]mazindol binding. Post-treatment with the inhibitors showed that the administration of nomifensine could be delayed longer than that of pargyline in order to achieve a protective effect. The results are compatible with the view that the monoamine oxidase-catalyzed conversion of MPTP to a toxic metabolite MPP + occurs mainly extraneuronally. MPP + is subsequently taken up and accumulated selectively in catecholamine neurons, initiating degeneration.

UDP-glucuronyltransferases and their toxicological significance.

In general chemicals are converted by phase-I enzymes of drug metabolism to a variety of nucleophilic and electrophilic metabolites (Fig. 1). The interaction of the more reactive electrophilic metabolites with critical cellular macromolecules plays a major role in their toxicity (Miller and Miller, 1981). Therefore much interest was given to the control of electrophilic metabolites. However the more stable and more abundant nucleophilic metabolites can also be readily converted to reactive metabolites. For example studies on benzene toxicity have shown that the major primary metabolite phenol is further oxidized to quinols, catechol and hydroquinone, in liver. These quinols are accumulating in bone marrow where they may be further oxidized to radical intermediates (Greenlee et al., 1981). Similarly, benzo(a)pyrene (BP) quinols are readily converted to reactive semiquinones. Moreover they undergo toxic redox-cycles between quinones and quinols with a continuous generation of reactive oxygen species (Lorentzen and Ts’o, 1977; Lorentzen et al., 1979; Lilienblum et al., 1985). Therefore the control of nucleophilic metabolites by UDP-glucuronyltransferase (GT) and sulfotransferase cannot be neglected. In the case of benzene toxicity conjugation leads to the elimination of quinols. In support of this observation the time course of induction of UDP-glucuronyltransferase (GT) by 3,4,3′,4′-tetrachlorobiphenyl mirrored the time course of protection against benzene toxicity in the bone marrow and lymphoid organs (Greenlee and Irons, 1981). In this context it is to be noted that some conjugates, in particular sulfate esters, are more reactive than the parent compounds (Miller and Miller, 1978).KeywordsLiver MicrosomeLiver NoduleArylhydrocarbon HydroxylaseToxicological SignificanceGlucuronide FormationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The time course of protection from delayed neurotoxicity induced by tri- o-cresyl phosphate and o, o-diisopropyl phosphorofluoridate by phenyl methyl sulfonyl fluoride in chickens

The time dependence of the ability of phenyl methyl sulfonyl fluoride (PMSF) to protect adult hens from developing signs of paralysis following the administration of 750 mg/kg tri- ortho-cresyl phosphate (TOCP), p.o., of 1.7 mg/kg O, O-diisopropyl phosphorofluoridate (DFP), s.c., was investigated. PMSF was able to protect the hens from organophosphorus-induced delayed neurotoxicity (OPIDN) when given between 1 and 24 h before the administration of TOCP, or when given 4 h before DFP. However, PMSF was ineffective at preventing paralysis when given 24 h following dosing with TOCP or when given later than 4 h before DFP administration. These results support the notion that PMSF acts at the same site as the organophosphorus esters.

Time course of protection from audiogenic seizures by glucose and insulin in audiogenically primed C57BL/6J mice

Glucose and insulin injections both decrease seizure susceptibility in C57BL/6J mice subjected to audiogenic priming at 16 days of age and tested at 21 days of age. These data support an hypothesis of a defect in immediately available energy reserves in brains of audiogenic seizure prone mice.

Presynaptic inhibition: the mechanism of protection from habituation of the crayfish lateral giant fibre escape response.

1. Mechanism of protection from habituation of the lateral giant escape reflex of the crayfish was studied. Experiments were designed to determine whether presynaptic inhibition of primary afferents for the reflex occurs following escape command neurone firing, and if so, whether it could account for protection of the first synapse from depression. 2. Synaptic transmission between afferents and interneurone A of the escape reflex is strongly inhibited following giant fibre spikes. 3. Giant fibre firing results in post-synaptic inhibition of interneurone A. However, inhibition of afferent input to interneurone A consistently outlasts both i.p.s.p.s and post-synaptic conductance increases in the neurone; the inhibition, therefore, is probably not exclusively post-synaptic. 4. Giant fibre firing results in excitability changes in sensory afferent terminals as measured by the amplitude of antidromic compound action potentials to focal electrical stimuli applied in the region of afferent terminals in the last abdominal ganglion. The time course of this effect parallels those of protection and inhibition of the first synapse. 5. The magnitude and time course of protection and inhibition of transmission to interneurone A parallel each other closely. Both processes considerably outlast measurable signs of post-synaptic inhibition. 6. We conclude that following giant fibre activity the first synapse of the lateral giant reflex is presynaptically inhibited and the presynaptic inhibition is responsible for the protection effect described in the preceding paper.

Time course of protection by N'N--diphenyl-p-phenylendiamine (DPPD) against carbon tetrachloride hepatotoxicy.

A preliminary treatment with DPPD (N,N'-diphenyl-p-phenylendiamine) prevents liver fatty infiltration early after carbon tetrachloride poisoning. Double bond shifting in microsomal lipids appears to be depressed 30 minutes after intoxication. A more active protection takes place when low doses of CCl4 are given (25 μl per 100 g body weight) than after a large dose of the poison (250 μl). In later stages of the halogenoalkane challenge, both triglyceride accumulation and lipid peroxidation take place despite of the antioxidant treatment. However, they are less severe and more transient than in unprotected animals. Similarly, liver necrosis, as evidenced by changes in serum activity of alanine aminotransferase appears, to be prevented by DPPD. This compound does not interfere with the hepatic metabolism of carbon tetrachloride, as suggested by the findings of14C binding to liver lipids and exhalation of radioactive CO2 after administration of14CCl4.

The Distribution and Metabolism of the Radiation Protective Agent Aminopentylaminoethylphosphorothioate in Mice

Abstract : Aminopropylaminoethyl-phosphorothioate (WR-2721) is the least toxic and most effective of the phosphorothioate class of radiation protective agents. In order to investigate the mechanism of action of this agent, distribution and metabolism studies were carried out using 35S-labelled WR-2721. In mice given WR-2721 intraperitoneally, tissue analysis showed only the unchanged compound and the protective agent bound to proteins by mixed disulfide bond. Concentration of the latter form correlated best with the time course of protection. Dephosphorylation to yield the thiol form of WR-2721, necessary for mixed disulfide formation on tissue proteins, was best demonstrated in the kidneys, both in vivo and in vitro, but also occurs in other tissues. Preliminary studies with the disulfide and thiol forms of WR-2721, which are less protective and more toxic than the phosphorothioate, suggest that the presence of the phosphate is necessary to mask the sulfhydryl group during transport to the target site. Formation of a mixed disulfide with tissue protein sulfhydryl groups at the target site appears to yield the final necessary condition for radiation protection. (Author)