Biomedical subjects

Jian Liu

Publications and source records attributed to Jian Liu.

At least 145 records · Page 8Linked to original sources

PubMed2004

Toxicity of familial ALS-linked SOD1 mutants from selective recruitment to spinal mitochondria.

One cause of amyotrophic lateral sclerosis (ALS) is mutation in ubiquitously expressed copper/zinc superoxide dismutase (SOD1), but the mechanism of toxicity to motor neurons is unknown. Multiple disease-causing mutants, but not wild-type SOD1, are now demonstrated to be recruited to mitochondria, but only in affected tissues. This is independent of the copper chaperone for SOD1 and dismutase activity. Highly preferential association with spinal cord mitochondria is seen in human ALS for a mutant SOD1 that accumulates only to trace cytoplasmic levels. Despite variable proportions that are successfully imported, nearly constant amounts of SOD1 mutants and covalently damaged adducts of them accumulate as apparent import intermediates and/or are tightly aggregated or crosslinked onto integral membrane components on the cytoplasmic face of those mitochondria. These findings implicate damage from action of spinal cord-specific factors that recruit mutant SOD1 to spinal mitochondria as the basis for their selective toxicity in ALS.

Aging↗

PubMed2004

Electrospray ionization with a pointed carbon fiber emitter.

A new type of electrospray ionization emitter employing a pointed carbon fiber has been developed for interfacing nanoliquid sampling techniques to mass spectrometry. The pointed carbon fiber protruding from an orifice with a surrounding hydrophobic surface confines a small Taylor cone at the tip, which generates a stable electrospray at the tip point. The small Taylor cone improves the electrospray efficiency thereby enhancing the detection limit. This emitter is rugged and able to generate stable electrospray over a wide range of flow rate, ESI voltage, and surface tension variation. Using a solution of angiotensin I, the carbon fiber emitter in 75-microm-i.d. fused-silica tubing was shown to give ion current comparable to that from a commercial 8 microm orifice nanospray emitter. Use of the emitter for ESI-MS/MS analysis of peptides was examined by infusing a mixture of cytochrome c and myoglobin tryptic digest peptides. Protein identification was demonstrated at the level of less than 1 fmol of the peptide consumed. The use of the carbon fiber emitter for interfacing monolithic capillary HPLC to MS was also demonstrated.

Animals↗

PubMed2004

Analysis of the interaction between adeno-associated virus and heparan sulfate using atomic force microscopy.

Adeno-associated virus (AAV) has been widely used as a viral vector to deliver genes to animal and human tissues in gene therapy studies. Both AAV-2 and AAV-3 use cell surface heparan sulfate (HS), a highly sulfated polysaccharide, as a receptor to establish infections. In this study, we used atomic force microscopy (AFM) to investigate the interaction of HS and AAV. A silicon chip functionalized with HS was used as a substrate for binding AAV for AFM analysis. To validate our approach, we found that the binding of AAV-2 to the HS surface was effectively competed by soluble HS, suggesting that the binding of AAV-2 to the functionalized surface was specific. In addition, we examined the binding of various AAV serotypes, including AAV-1, AAV-2, AAV-3, and AAV-5, to the HS surface. As expected, only AAV-2 and AAV-3 bound, whereas AAV-1 and AAV-5 did not. This observation was consistent with the previous conclusion that AAV-1 and AAV-5 do not use HS as a receptor for infection. In conclusion, we developed a novel approach to investigate the interaction of AAV virus with its polysaccharide-based receptor at the level of a single viral particle. Given that HSs serve as receptor for numerous viruses, this approach has the potential to become a generalized method for studying interactions between the viral particle and HS, as well as other virus-cell interactions, and potentially serve as a platform for screening antiviral therapies.

Cell Line↗

PubMed2004

Lysosomal cysteine proteases in atherosclerosis.

Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis. Atherosclerotic lesions in humans overexpress the elastolytic and collagenolytic cathepsins S, K, and L but show relatively reduced expression of cystatin C, their endogenous inhibitor, suggesting a shift in the balance between cysteine proteases and their inhibitor that favors remodeling of the vascular wall. Extracts of human atheromatous tissue show greater elastolytic activity in vitro than do those from healthy donors. The cysteinyl protease inhibitor E64d limits this increased elastolysis, indicating involvement of cysteine proteases in elastin degradation during atherogenesis. Furthermore, inflammatory cytokines augment expression and secretion of active cysteine proteases from cultured monocyte-derived macrophages, vascular smooth muscle cells, and endothelial cells and increase degradation of extracellular elastin and collagen. Cathepsin S-deficient cells or those treated with E64d show significantly impaired elastolytic or collagenolytic activity. Additionally, recent in vivo studies of atherosclerosis-prone, LDL receptor-null mice lacking cathepsin S show participation of this enzyme in the initial infiltration of leukocytes, medial elastic lamina degradation, endothelial cell invasion, and neovascularization, illustrating an important role for cysteine proteases in arterial remodeling and atherogenesis.

Animals↗

PubMed2004

Daily short-period gravitation can prevent functional and structural changes in arteries of simulated microgravity rats.

This study was designed to clarify whether simulated microgravity-induced differential adaptational changes in cerebral and hindlimb arteries could be prevented by daily short-period restoration of the normal distribution of transmural pressure across arterial vasculature by either dorsoventral or footward gravitational loading. Tail suspension (Sus) for 28 days was used to simulate cardiovascular deconditioning due to microgravity. Daily standing (STD) for 1, 2, or 4 h, or +45 degrees head-up tilt (HUT) for 2 or 4 h was used to provide short-period dorsoventral or footward gravitational loading as countermeasure. Functional studies showed that Sus alone induced an enhancement and depression in vasoconstrictor responsiveness of basilar and femoral arterial rings, respectively, as previously reported. These differential functional alterations can be prevented by either of the two kinds of daily gravitational loading treatments. Surprisingly, daily STD for as short as 1 h was sufficient to prevent the differential functional changes that might occur due to Sus alone. In morphological studies, the effectiveness of daily 4-h HUT or 1-h STD in preventing the differential remodeling changes in the structure of basilar and anterior tibial arteries induced by Sus alone was examined by histomorphometry. The results showed that both the hypertrophic and atrophic changes that might occur, respectively, in cerebral and hindlimb arteries due to Sus alone were prevented not only by daily HUT for 4 h but also by daily STD even for 1 h. These data indicate that daily gravitational loading by STD for as short as 1 h is sufficient to prevent differential adaptational changes in function and structure of vessels in different anatomic regions induced by a medium-term simulated microgravity.

Adaptation, Physiological↗

PubMed2004

A conformational change in heparan sulfate 3-O-sulfotransferase-1 is induced by binding to heparan sulfate.

The 3-O-sulfation of glucosamine by heparan sulfate 3-O-sulfotransferase-1 (3-OST-1) is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). In this paper, we present evidence of a conformational change that occurs in 3-OST-1 upon binding to heparan sulfate. The intrinsic fluorescence of 3-OST-1 was increased in the presence of HS, suggesting a conformational change. This apparent conformational change was further investigated using differential chemical modification of 3-OST-1 to measure the solvent accessibility of the lysine residues. 3-OST-1 was treated with acetic anhydride in either the presence or absence of HS using both acetic anhydride and hexadeuterioacetic anhydride under nondenaturing and denaturing conditions, respectively. The relative reactivity of the lysine residues to acetylation and [2H] acetylation in the presence or absence of HS was analyzed by measuring the ratio of acetylated and deuterioacetylated peptides using matrix-assisted laser desorption ionization mass spectrometry. The solvent accessibilities of the lysine residues were altered differentially depending on their location. In particular, we observed a group of lysine residues in the C-terminus of 3-OST-1 that become more solvent accessible when 3-OST-1 binds to HS. This observation indicates that a conformational change could be occurring during substrate binding. A truncated mutant of 3-OST-1 that lacked this C-terminal region was expressed and found to exhibit a 200-fold reduction in sulfotransferase activity. The results from this study will contribute to our understanding of the interactions between 3-OSTs and HS.

Amino Acid Substitution↗

PubMed2004

Crystal structure and mutational analysis of heparan sulfate 3-O-sulfotransferase isoform 1.

Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO(3)) from 3'-phosphoadenosine 5'-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate. This structure reveals residues critical for 3'-phosphoadenosine 5'-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases.

Amino Acid Sequence↗

PubMed2004

The biosynthesis of anticoagulant heparan sulfate by the heparan sulfate 3-O-sulfotransferase isoform 5.

The role of heparan sulfate (HS) in regulating blood coagulation has a wide range of clinical implications. In this study, we investigated the role of 3-O-sulfotransferase isoform 5 (3-OST-5) in generating anticoagulant HS in vivo. A Chinese hamster ovary cell line (3OST5/CHO) stably expressing 3-OST-5 was generated. The expression of 3-OST-5 in 3OST5/CHO cells was confirmed by Northern blot analysis, RT-PCR, and the disaccharide analyses of the HS from the cells. We also determined the effects of the HS from 3OST5/CHO on antithrombin-mediated inhibition of factor Xa. Fluorescently labeled antithrombin bound to the surface of 3OST5/CHO cells, suggesting that the antithrombin-binding HS is indeed present on the cell surface. Our results demonstrate that the 3-OST-5 gene is capable of synthesizing anticoagulant HS in CHO cells and has the potential to contribute to the biosynthesis of anticoagulant HS in humans.

Animals↗

PubMed2004

Conditioning processes contribute to severity of naloxone-precipitated withdrawal from acute opioid dependence.

RATIONALE: Single injections with morphine can induce a state of acute opioid dependence in humans and animals, typically measured as precipitated withdrawal when an antagonist such as naloxone is administered 4-24 h after morphine. Repeated treatment with morphine results in further increases in naloxone potency, and prior work has shown that this progressive shift in naloxone potency requires repeated naloxone experience under some but not all experimental conditions. OBJECTIVE: The current study sought to further characterize the experimental conditions that support naloxone experience-dependent and experience-independent potentiation of precipitated suppression of operant responding in morphine pretreated rats, and to assess more directly whether conditioning mechanisms may contribute to the former process. METHODS: Rats trained on an FR15 schedule for food received a total of five vehicle or morphine injections (5.6 mg/kg SC) at 4, 8, or 22 h prior to an operant session in which a cumulative dose-effect function for naloxone-induced suppression of responding was determined. Separate groups of animals at each interval between morphine and naloxone received cumulative naloxone dosing after all morphine pretreatments (NAL ALL DAYS) or after just the first and last morphine pretreatment (NAL FIRST/LAST). Additional groups of rats at the 4 h MOR-NAL interval received most of their naloxone cumulative dose-effect experience in either the home cage or in the operant context with levers retracted. RESULTS: Vehicle-pretreated (Morphine-Naive) rats showed little change in the naloxone dose-effect function even after five cumulative dose-effect determinations. With a single morphine pretreatment, naloxone potency was increased at 4 or 8 h post-morphine, but not at 22 h. With repeated morphine treatment, all MOR-NAL intervals resulted in significant shifts in naloxone potency across treatment days even when naloxone was administered only after the first and last morphine pretreatment. However, much greater shifts in naloxone potency were observed at 4-h and 8-h intervals when naloxone was administered on all treatment days. At the 22 h MOR-NAL interval, there was no further potentiation in naloxone potency with additional naloxone experience provided on the intermediate days. Finally, when the repeated naloxone experience occurred in the home cage at the 4-h interval, naloxone potency was identical to that seen after limited naloxone experience (NAL FIRST/LAST), and significantly less than naloxone potency in groups receiving repeated naloxone experience in the operant context. CONCLUSIONS: The results suggest that conditioned withdrawal mechanisms may play a significant role in the initial development of opioid dependence.

Animals↗

PubMed2004

ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense.

The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness. In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture. The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox. A variety of clones were evaluated, and many were unacceptably virulent in animal models. One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells. ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates. To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions. The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes. ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials. The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax.

Animals↗

PubMed2004

Isocyanate vapor-induced antigenicity of human albumin.

BACKGROUND: The bioreactivity of isocyanate, a leading cause of occupational asthma, has led to uncertainty regarding the chemical's antigenicity and mechanisms that elicit immunopathology. OBJECTIVE: To understand better the biologically relevant antigenic forms of hexamethylene diisocyanate (HDI), commonly used in the auto body industry. METHODS: Human albumin was exposed to HDI vapors through a novel approach designed to model the air-liquid interface of the human airway. Vapor HDI-exposed albumin was characterized by electrophoresis, chemical substitution analysis, mass spectrometry, and serology studies on auto body shop workers (N=203) and HDI asthmatics (N=11). RESULTS: HDI vapors caused significant changes in the shape and/or charge of human albumin, which differed from albumin exposed to liquid phase HDI, with lower isocyanate substitution ratios and distinct electrophoretic mobility. Specific sites of vapor HDI conjugation to albumin were identified at His(247) and Lys(414). Vapor HDI-exposed albumin was specifically recognized by the humoral arm of the human immune system, with a strong dependence on albumin as the carrier. Vapor HDI-exposed albumin-specific IgG titers were significantly associated with HDI exposure (P=.001), and specific IgE was detectable in 55% (6/11) of isocyanate asthmatics versus 1.5% (3/203) of exposed healthy workers. Parallel studies using HDI-exposed albumin conjugates produced by previously published methods showed less significant associations of HDI-specific IgG and IgE with exposure and disease, respectively. CONCLUSION: HDI-albumin conjugates produced by novel vapor phase exposure methods may be more physiologically relevant than those produced by previously published methods and of greater utility in characterizing the immune responses associated with HDI exposure and asthma.

Albumins↗

PubMed2004

Brain reward deficits accompany naloxone-precipitated withdrawal from acute opioid dependence.

Single injections with morphine can induce a state of acute opioid dependence in humans and animals, typically measured as precipitated withdrawal when an antagonist such as naloxone is administered 4-24 h after morphine. Repeated treatment with morphine results in a progressive shift in potency of naloxone to produce such acute withdrawal signs. The current study examined alterations in brain reward thresholds after acute and repeated treatment with morphine (5.6 mg/kg) using a discrete-trial current-intensity brain-stimulation reward procedure. Rats with stimulation electrodes aimed at the medial forebrain bundle at the level of the lateral hypothalamus were tested in twice daily sessions separated by 4 h. Separate groups of rats received treatment with morphine immediately after the first daily test session, and one of several doses of naloxone (0.10, 0.33, 1.0 mg/kg) 4 h later and immediately before the second session; these morphine and naloxone treatments were repeated for four consecutive days (Morphine-Repeat NAL). Additional groups examined the independent contribution of repeated morphine or repeated naloxone. One control group (Morphine-Vehicle) received morphine on all four treatment days, but vehicle before the second test session. A second group (Morphine-Single NAL) also received morphine on all four treatment days, but received 1.0 mg/kg only once after the final morphine pretreatment. A final control group received no morphine at all but received the 1.0-mg/kg dose of naloxone four times (Vehicle-Repeat NAL) before the second daily test session. Repeated naloxone alone (Vehicle-Repeat NAL) produced no changes in brain reward thresholds. Repeated morphine alone (Morphine-Vehicle) failed to alter reward thresholds measured 4 h postmorphine, but produced a slight increase in thresholds in the test sessions that occurred before morphine treatment on Days 3 and 4 (and hence 23.5 h after the previous day's morphine injection). This suggested the development of a modest spontaneous withdrawal-induced reward deficit measurable at 23.5 but not 4 h postmorphine. Naloxone dose-dependently increased brain reward thresholds 4 h after a single morphine pretreatment, with a further shift to the left in the naloxone dose-effect function resulting from repeated morphine and naloxone administration (Morphine-Repeat NAL). However, when the highest dose of naloxone was tested only after the final morphine pretreatment (Morphine-Single NAL), its potency was no different than when administered after the first morphine pretreatment. The results indicate that neuroadaptation within brain reward circuitry results in significant reward deficits after a single morphine pretreatment, and this deficit increases rapidly with repeated morphine and naloxone-induced withdrawal experience.

Acute Disease↗

PubMed2004

Morpho-regulation of ectodermal organs: integument pathology and phenotypic variations in K14-Noggin engineered mice through modulation of bone morphogenic protein pathway.

Ectodermal organs are composed of keratinocytes organized in different ways during induction, morphogenesis, differentiation, and regenerative stages. We hypothesize that an imbalance of fundamental signaling pathways should affect multiple ectodermal organs in a spatio-temporal-dependent manner. We produced a K14-Noggin transgenic mouse to modulate bone morphogenic protein (BMP) activity and test the extent of this hypothesis. We observed thickened skin epidermis, increased hair density, altered hair types, faster anagen re-entry, and formation of compound vibrissa follicles. The eyelid opening was smaller and ectopic cilia formed at the expense of Meibomian glands. In the distal limb, there were agenesis and hyperpigmentation of claws, interdigital webbing, reduced footpads, and trans-differentiation of sweat glands into hairs. The size of external genitalia increased in both sexes, but they remained fertile. We conclude that modulation of BMP activity can affect the number of ectodermal organs by acting during induction stages, influence the size and shape by acting during morphogenesis stages, change phenotypes by acting during differentiation stages, and facilitate new growth by acting during regeneration stages. Therefore during organogenesis, BMP antagonists can produce a spectrum of phenotypes in a stage-dependent manner by adjusting the level of BMP activity. The distinction between phenotypic variations and pathological changes is discussed.

Animals↗

PubMed2004

Molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, Sulfolobus shibatae DSM5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme.

A gene coding for an esterase (SshEstI, 915 bp in length) of the thermoacidophilic archaeon Sulfolobus shibatae DSM5389 was cloned, sequenced, and overexpressed in Escherichia coli JM109 cells as a soluble, catalytically active protein. The deduced amino acid sequence of SshEstI was consistent with a protein containing 305 amino acid residues with a molecular mass of 33 kDa. Sequence comparison studies indicated that SshEstI could be a member of the hormone-sensitive lipase family, in that it had the highest sequence similarity to esterases from Sulfolobus solfataricus (90% identity) and Archaeoglobus fulgidus (42%) and a lipase from Pseudomonas sp. B11-1 (38%). The recombinant enzyme was highly thermostable and retained more than 70% of its initial activity after incubation at 90 degrees C and pH 7.0 for 30 min. The recombinant enzyme catalyzed the hydrolysis of p-nitrophenyl (p-NP) esters with C2-C16 acyl chains but not the hydrolysis of triacylglycerides such as tributyrin and triolein. The enzymatic hydrolysis of p-NP acetate proceeded in a linear manner with time, whereas that of p-NP esters with acyl chains of C5 or longer showed a biphasic profile, where a rapid release of p-nitrophenol ( approximately 3 min) was followed by a slow, sustained release. These non-linear kinetics may be explained in terms of a very slow, presteady-state burst phenomenon of p-nitrophenol release or a hysteretic behavior of SshEstI with these substrates.

Journal Article↗

PubMed2004

Transfer of a gene containing the Arg-Gly-Asp peptide prolongs the bleeding time of mice.

Peptides containing Arg-Gly-Asp (RGD) have been used to decrease thrombosis by competitive inhibition of the integrin glycoprotein, alphaIIb/beta3a, in platelets. However, they have a short half-life in vivo. A naked plasmid, pCMV-RGD, was transferred into the skeletal muscle of mice and RGD gene expression was observed by RT-PCR. The bleeding time between control mice and RGD-transferred mice was prolonged from the 10th day to the 80th day after gene transfer while the blood glucose and serum insulin-like proteins remained at normal levels. These results provided a convenient and effective approach to relieve patients from thrombi in a single step over a relatively long period.

Animals↗

PubMed2004

A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1.

Membrane fusion induced by herpes simplex virus (HSV) is required for both entry and cell-to-cell spread. It is mediated by the viral glycoprotein gB, gD, gH-gL and gD receptors. Although 3-O-sulfated heparan sulfate (3-OS HS) is a receptor for HSV-1 entry, the requirement for heparan sulfate in the fusion process has been ruled out. Here, it is demonstrated that cells expressing 3-OS HS, generated by D-glucosaminyl 3-O-sulfotransferase isoforms-3 and/or -5 (3-OST-3 and 3-OST-5), fused with cells expressing the four glycoproteins. The cell fusion observed exhibited similar requirements but was independent of protein receptors, HVEM or nectin-1. Additionally, removal of 3-OS HS from the cell surface by heparinase-I treatment and, in separate experiments, the presence of soluble 3-OST-3- and 3-OST-5-modified HS, significantly inhibited fusion. Taken together, these results indicate that 3-OS HS can play a crucial role in virus entry and cell fusion.

Animals↗

PubMed2004

Serine-threonine kinases and transcription factors active in signal transduction are detected at high levels of phosphorylation during mitosis in preimplantation embryos and trophoblast stem cells.

Serine-threonine kinases and transcription factors play important roles in the G1-S phase progression of the cell cycle. Assays that use quantitative fluorescence by immunocytochemical means, or that measure band strength during Western blot analysis, may have confused interpretations if the intention is to measure G1-S phase commitment of a small subpopulation of phosphorylated proteins, when a larger conversion of the same population of proteins can occur during late G2 and M phases. In mouse trophoblast stem cells (TSC), a human placental cell line (HTR), and/or mouse preimplantation embryos, 8/19 serine-threonine and tyrosine kinases, 3/8 transcription factors, and 8/14 phospho substrate and miscellaneous proteins were phosphorylated at higher levels in M phase than in interphase. Most phosphoproteins appeared to associate with the spindle complex during M phase, but one (p38MAPK) associated with the spindle pole and five (Cdx2, MEK1, 2, p27, and RSK1) associated with the DNA. Phosphorylation was detected throughout apparent metaphase, anaphase and telophase for some proteins, or for only one of these segments for others. The phosphorylation was from 2.1- to 6.2-fold higher during M phase compared with interphase. These data suggest that, when planning and interpreting quantitative data and perturbation experiments, consideration must be given to the role of serine-threonine kinases and transcription factors during decision making in M phase as well as in G1-S phase.

Animals↗

PubMed2004

ROS-mediated ERK activation in delayed protection from anoxic preconditioning in neonatal rat cardiomyocytes.

BACKGROUND: The activation of extracellular signal-regulated kinase1/2 (ERK1/2) has been shown to be important signaling pathway in the ischemic preconditioning (IPC) response. Recently, some studies suggest a key role for the mitochondrial ATP-sensitive potassium channel (mKATP) as both a trigger and an end effector of acute and delayed protection of IPC. Hence, this study was undertaken to elucidate the relationship between mKATP and ERK1/2 in the delayed protection mechanism of anoxic preconditioning (APC). METHODS: An APC model was established using cultured neonatal rat cardiomyocytes. Pharmacological agents [diazoxide, 5-hydroxydecanoate (5-HD), 2-mercaptopropionylglycine (MPG), and PD98059] were used to modulate mKATP and ERK1/2 activation. Cellular injury was evaluated by measuring cellular superoxide dismutase (SOD) activity, cell viability, and lactate dehydrogenase (LDH) release. The generation of cellular reactive oxygen species (ROS) and the activation of ERK1/2 were determined at different time points starting from the beginning of preconditioning with anoxia or diazoxide (an mKATP opener). RESULTS: Cell viability and SOD activity in the APC [(81.9 +/- 11.4)%, (13.6 +/- 3.7) U/L] and diazoxide [(79.2 +/- 12.4)%, (16.5 +/- 4.6) U/L] groups were significantly higher than in the anoxia/reoxygenation (A/R) [(42.2 +/- 7.3)%, (8.8 +/- 2.8) U/L] group (all P < 0.01). LDH activity in the APC group [(101.9 +/- 18.9) U/L] and diazoxide group [(97.5 +/- 17.7) U/L] was significantly lower than in the A/R group [(250.5 +/- 43.6) U/L] (all P < 0.01). Both APC and diazoxide simultaneously facilitated intracellular ROS generation and rapid ERK1/2 activation. But the effects of APC and diazoxide were remarkedly attenuated by 5-HP (an mKATP blocker) and by MPG (a free radical scavenger). In addition, the ERK1/2 inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide. CONCLUSION: mKATP may mediate ERK1/2 activation during anoxia preconditioning by generating ROS, which then triggers the delayed protection of APC in rat cardiomyocytes.

Animals↗