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Biomedical subjects

M Chen

Publications and source records attributed to M Chen.

At least 109 records · Page 6Linked to original sources
PubMed2001

Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion.

Reperfusion after myocardial ischemia is associated with a rapid influx of calcium, leading to activation of various enzymes including calpain. Isolated perfused adult rabbit hearts subjected to global ischemia and reperfusion were studied. Calpain or a calpain-like activity was activated within 15 min after reperfusion, and preconditioning suppressed calpain activation. In contrast, caspase activation was not detected although cytochrome c was released after ischemia and reperfusion. The pro-apoptotic BH3-only Bcl-2 family member, Bid, was cleaved during ischemia/reperfusion in the adult rabbit heart. Recombinant Bid was cleaved by calpain to a fragment that was able to mediate cytochrome c release. The calpain cleavage site was mapped to a region within Bid that is extremely susceptible to proteolysis. These findings suggest that there is cross-talk between apoptotic and necrotic pathways in myocardial ischemia/reperfusion injury.

Amino Acid Sequence↗
PubMed2001

Liposome-mediated gene transfection of endothelial nitric oxide synthase reduces endothelial activation and leukocyte infiltration in transplanted hearts.

BACKGROUND: During cardiac ischemia-reperfusion injury, neutrophilic infiltration of the myocardium is mediated by adhesion molecule expression on activated coronary endothelium. Nitric oxide inhibits neutrophil adhesion to endothelium in vitro by blocking the nuclear factor (NF)-kappaB-dependent transcription of adhesion molecules. We investigated whether intraoperative gene delivery of endothelial nitric oxide synthase (eNOS) into donor hearts before transplantation would have a similar effect on an entire organ. METHODS AND RESULTS: In an allogeneic rabbit heart transplant model, liposomes complexed to the gene encoding eNOS were infused into the donor coronary circulation before transplantation. By 24 hours after transplantation, calcium-dependent nitrite production was significantly higher in eNOS-transfected donor hearts than in the 3 control groups: donor hearts transfected with empty plasmids alone, donor hearts treated with diluent only, and untransplanted native hearts. Intramyocardial neutrophil and T-lymphocyte populations were halved in eNOS-transfected hearts compared with control donor hearts (P<0.05). Moreover, the prevalence of NF-kappaB activation in microvascular endothelial cells and surrounding cardiac myocytes as well as endothelial vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 expression were all significantly reduced in eNOS-transfected hearts compared with control donor hearts (P<0.01). Without immunosuppression, eNOS-transfected hearts survived longer than controls. CONCLUSIONS: Intraoperative liposome-mediated gene delivery of eNOS to donor hearts can result in early gene expression sufficient to reduce ischemia-reperfusion injury by inhibiting NF-kappaB activation, adhesion molecule expression, and the early infiltration of leukocytes, all of which may improve graft survival.

Animals↗
PubMed2001

Reversal of spontaneous autoimmune insulitis in nonobese diabetic mice by soluble lymphotoxin receptor.

One striking feature of spontaneous autoimmune diabetes is the prototypic formation of lymphoid follicular structures within the pancreas. Lymphotoxin (LT) has been shown to play an important role in the formation of lymphoid follicles in the spleen. To explore the potential role of LT-mediated microenvironment in the pathogenesis of insulin-dependent diabetes mellitus (IDDM), an LTbeta receptor-immunoglobulin fusion protein (LTbetaR-Ig) was administered to nonobese diabetic mice. Early treatment with LTbetaR-Ig prevented insulitis and IDDM, suggesting that LT plays a critical role in the insulitis development. LTbetaR-Ig treatment at a late stage of the disease also dramatically reversed insulitis and prevented diabetes. Moreover, LTbetaR-Ig treatment prevented the development of IDDM by diabetogenic T cells in an adoptive transfer model. Thus, LTbetaR-Ig can disassemble the well established lymphoid microenvironment in the islets, which is required for the development and progression of IDDM.

Animals↗
PubMed2001

A selenium-containing catalytic antibody with Type I deiodinase activity.

Acting as a mimic of type I deiodinase (DI), a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against thyroxine (T4) into selenocysteines, can catalyze the deiodination of T(4) to 3,5,3'-triiodothyronine (T(3)) with dithiothreitol (DTT) as cosubstrate. Investigations into the deiodinative reaction by Se-4C5 revealed the relationship between the initial velocity and substrate concentration was subjected to Michaelis-Menten equation and the reaction mechanism was ping-pong one. The kinetic properties of the catalytic antibody were a little similar to those of DI, with Km values for T(4) and DTT of approximately 0.8 microM and 1.8 mM, respectively, and V(m) value of 270 pmol per mg protein per min. The activity could be sensitively inhibited by PTU with a Ki value of approximately 120 microM at 2.0 microM of T(4) concentration, revealing that PTU was a competitive inhibitor for DTT.

Amino Acid Substitution↗
PubMed2001

ADAR1 is involved in the development of microvascular lung injury.

Deamination of adenosine on pre-mRNA to inosine is a recently discovered process of posttranscription modification of pre-mRNA, termed A-to-I RNA editing, which results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the development of microvascular lung injury. To that end, the pulmonary expression and activity of the RNA editase ADAR1 were evaluated in a mouse model of endotoxin (15 mg/kg IP)-induced microvascular lung injury (n=5) as well as in cultured alveolar macrophages stimulated with endotoxin, live bacteria, or interferon. ADAR1 expression and activity were identified in sham lungs that were upregulated in lungs from endotoxin-treated mice (at 2 hours). Expression was localized to polymorphonuclear and monocytic cells. These events preceded the development of pulmonary edema and leukocyte accumulation in lung tissue and followed the local production of interferon-gamma, a known inducer of ADAR1 in other cell systems. ADAR1 was found to be upregulated in alveolar macrophages (MH-S cells) stimulated with endotoxin (1 to 100 microg/mL), live Escherichia coli (5x10(7) colony-forming units), or interferon-gamma (1000 U/mL). Taken together, these data suggest that ADAR1 may play a role in the pathogenesis of microvascular lung injury possibly through induction by interferon.

Adenosine↗
PubMed2001

[Construction of attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori catalase and observation on its protective immunity].

OBJECTIVE: To investigated the effect of attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori (Hp) catalase (KatA) on protection against Hp infection. METHODS: Recombinant plasmid expressing KatA was constructed. Expression of KatA was induced with IPTG, and analyzed by SDS-PAGE and Western blot. The recombinant plasmid were introduced into attenuated Salmonella typhimurium SL3261 strain to construct live oral vaccine strain. C57BL/6 mice was orally immunized with this vaccine strain and then given orogastric challenge with live Hp Sydney strain. The stomach samples were submitted to a rapid urease test and quantitative culture. RESULTS: The SDS-PAGE showed a dominant additional protein with molecular weight of 79kDa which could specially react with antibody against GST and accounted for 19% of total bacteria proteins. Animal experiment indicated that this vaccine strain could protect mice against Hp infection. CONCLUSION: Attenuated Salmonella typhimurium vaccine strain expressing KatA could induce effective immune response in protection against Hp infection,which may play an important role in preventing and treating Hp infection and related diseases.

Administration, Oral↗
PubMed2001

Preparation and properties of a selenium-containing catalytic antibody as type I deiodinase mimic.

Conversion of thyroxine (T4) to 3,5,3'-triiodothyronine is an essential first step in controlling thyroid hormone action. Type I deiodinase (DI) can catalyze the conversion to produce the bulk of serum 3,5,3'-triiodothyronine. Acting as a mimic of DI, a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against T4 into selenocysteines, can catalyze the deiodination of T4 with dithiothreitol (DTT) as cosubstrate. The mimic enzyme Se-4C5 exhibited a much greater deiodinase activity than model compound ebselen and another selenium-containing antibody Se-Hp4 against GSH. The coupling of selenocysteine with the combining pocket of antibody 4C5 endowed Se-4C5 with enzymatic activity. To probe the catalytic mechanism of the catalytic antibody, detailed kinetic studies were carried out in this paper. Investigations into the deiodinative reaction revealed the relationship between the initial velocity and substrate concentration. The characteristic parallel Dalziel plots demonstrated that Se-4C5-catalyzed reaction mechanism was ping-pong one, involving at least one covalent enzyme intermediate. The kinetic properties of the catalytic antibody were similar to those of DI, with Km values for T4 and DTT of approximately 0.8 microm and 1.8 mm, respectively, and a Vm value of 270 pmol per mg of protein per min. The activity could be sensitively inhibited by 6-propyl-2-thiouracil (PTU) with a K(i) value of approximately 120 microm at 2.0 microm T4 concentration. The PTU inhibition was progressively alleviated with the increasing concentration of added DTT, revealing that PTU was a competitive inhibitor for DTT.

Animals↗
PubMed2001

Conserved C-terminal residues within the lectin-like domain of LOX-1 are essential for oxidized low-density-lipoprotein binding.

Lectin-like oxidized low-density-lipoprotein (oxLDL) receptor-1 (LOX-1) is a cell-surface endocytosis receptor for atherogenic oxLDL, which is highly expressed in endothelial cells. Recent studies suggest that it may play significant roles in atherogenesis. LOX-1 is a type-II membrane protein that structurally belongs to the C-type lectin family molecules. This study was designed to characterize the specific domain on LOX-1 that recognizes oxLDL. Truncation of the lectin domain of LOX-1 abrogated oxLDL-binding activity. Deletion of the utmost C-terminal ten amino acid residues (261-270) was enough to disrupt the oxLDL-binding activity. Substitutions of Lys-262 and/or Lys-263 with Ala additively attenuated the activity. Serial-deletion analysis showed that residues up to 265 are required for the expression of minimal binding activity, although deletion of the C-terminal three residues (268-270) still retained full binding activity. Consistently, these alterations in LOX-1 impaired the recognition by a functionally blocking monoclonal antibody for LOX-1. These data demonstrated the distinct role of the lectin domain as the functional domain recognizing LOX-1 ligand. The conserved C-terminal residues of lectin-like domain are essential for binding oxLDL. Particularly, the basic amino acid pair is important for the binding.

Amino Acid Sequence↗
PubMed2001

Structure-based design of a bispecific receptor mimic that inhibits T cell responses to a superantigen.

Key surface proteins of pathogens and their toxins bind to the host cell receptors in a manner that is quite different from the way the natural ligands bind to the same receptors and direct normal cellular responses. Here we describe a novel strategy for "non-antibody-based" pathogen countermeasure by targeting the very same "alternative mode of host receptor binding" that the pathogen proteins exploit to cause infection and disease. We have chosen the Staphylococcus enterotoxin B (SEB) superantigen as a model pathogen protein to illustrate the principle and application of our strategy. SEB bypasses the normal route of antigen processing by binding as an intact protein to the complex formed by the MHC class II receptor on the antigen-presenting cell and the T cell receptor. This alternative mode of binding causes massive IL-2 release and T cell proliferation. A normally processed antigen requires all the domains of the receptor complex for its binding, whereas SEB requires only the alpha1 subunit (DRalpha) of the MHC class II receptor and the variable beta subunit (TCRVbeta) of the T cell receptor. This prompted us to design a bispecific chimera, DRalpha-linker-TCRVbeta, that acts as a receptor mimic and prevents the interaction of SEB with its host cell receptors. We have adopted (GSTAPPA)(2) as the linker sequence because it supports synergistic binding of DRalpha and TCRVbeta to SEB and thereby makes DRalpha-(GSTAPPA)(2)-TCRVbeta as effective an SEB binder as the native MHC class II-T cell receptor complex. Finally, we show that DRalpha-(GSTAPPA)(2)-TCRVbeta inhibits SEB-induced IL-2 release and T cell proliferation at nanomolar concentrations.

Amino Acid Sequence↗
PubMed2001

[Analgesic effect of neurolytic celiac plexus block guided by ultrasonography in advanced malignancies].

OBJECTIVE: To investigate the analgesic effect of celiac plexus block with anhydrous alcohol in patients suffering form advanced abdominal malignancies. METHODS: Ultrasonographically demonstrated and guided, 20 to 50 ml of anhydrous alcohol is injected into the celiac plexus and superior mesenteric plexus by fine needle of 22G. The injecting angle between the needle and the aorta is 20 to 30 degree. The injecting fields mainly located around the celiac plexus and the dorsal part of tumor are also injected. Sometimes if the tumor is rather large, an appropriate amount of alcohol was injected inside too. RESULTS: Twenty-eight cases of advanced abdominal malignancies were treated, among whom 96.4% achieved analgesic effect of different degree, 53.6% (15 cases) achieved the period of pain relief over three days, and 42.8% (12 cases) achieved partial relief. The analgesic effect is correlated with the amount of alcohol injected, i.e. no complete relief was observed in the four patients who received less than 12 ml, while two out of six could get transient complete relief in those who received 15 to 28 ml. In the group of 30 to 50 ml, 72.2% (13 cases) could get complete pain relief. Nine cases with big tumor received injection in side the tumor, with six tumors resulting in shrinkage or necrosis, significant pain relief also achieved in these cases. Complications occurred in nine cases (32.1%), with four cases of postural hypotension, two cases of vomiting, two cases of hiccup and one case of diarrhea. CONCLUSION: Neurolytic celiac plexus block guided by ultrasonography is a safe and effective modality in the management of intractable pain resulted from advanced abdominal malignancies.

Abdominal Neoplasms↗
PubMed2001

Self-replicative RNA vaccines elicit protection against influenza A virus, respiratory syncytial virus, and a tickborne encephalitis virus.

In genetic vaccination, recipients are immunized with antigen-encoding nucleic acid, usually DNA. This study addressed the possibility of using the recombinant alpha virus RNA molecule, which replicates in the cytoplasm of transfected cells, as a novel approach for genetic vaccination. Mice were immunized with recombinant Semliki Forest virus RNA-encoding envelope proteins from one of 3 viruses: influenza A virus, a tickborne flavivirus (louping ill virus), or respiratory syncytial virus (RSV). Serologic analyses showed that antigen-specific antibody responses were elicited. IgG isotyping indicated that predominantly Th1 type immune responses were induced after immunization with RSV F protein-encoding RNA, which is relevant for protection against RSV infection. Challenge infection showed that RNA immunization had elicited significant levels of protection against the 3 model virus diseases.

Animals↗
PubMed2001

Increased insulin sensitivity in Gsalpha knockout mice.

The stimulatory guanine nucleotide-binding protein (G(s)) is required for hormone-stimulated cAMP generation. Gnas, the gene encoding the G(s) alpha-subunit, is imprinted, and targeted disruption of this gene in mice leads to distinct phenotypes in heterozygotes depending on whether the maternal (m-/+) or paternal (+/p-) allele is mutated. Notably, m-/+ mice become obese, whereas +/p- mice are thinner than normal. In this study we show that despite these opposite changes in energy metabolism, both m-/+ and +/p- mice have greater sensitivity to insulin, with low to normal fasting glucose levels, low fasting insulin levels, improved glucose tolerance, and exaggerated hypoglycemic response to administered insulin. The combination of increased insulin sensitivity with obesity in m-/+ mice is unusual, because obesity is typically associated with insulin resistance. In skeletal muscles isolated from both m-/+ and +/p- mice, the basal rate of 2-deoxyglucose uptake was normal, whereas the rate of 2-deoxyglucose uptake in response to maximal insulin stimulation was significantly increased. The similar changes in muscle sensitivity to insulin in m-/+ and +/p- mice may reflect the fact that muscle G(s)alpha expression is reduced by approximately 50% in both groups of mice. GLUT4 expression is unaffected in muscles from +/p- mice. Increased responsiveness to insulin is therefore the result of altered insulin signaling and/or GLUT4 translocation. This is the first direct demonstration in a genetically altered in vivo model that G(s)-coupled pathways negatively regulate insulin signaling.

Animals↗
PubMed2001

The carboxyl terminus of type VII collagen mediates antiparallel dimer formation and constitutes a new antigenic epitope for epidermolysis Bullosa acquisita autoantibodies.

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC2 domain has been implicated in catalyzing the antiparallel dimer formation of type VII procollagen. In this study, we produced the entire 161 amino acids of the NC2 domain plus 186 amino acids of adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of collagen VII. Removal of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesis, we found that mutation of either cysteine 2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel dimer formation. In contrast, a single cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is associated with recessive dystrophic epidermolysis bullosa, was unable to form antiparallel dimers. Furthermore, autoantibodies from epidermolysis bullosa acquisita patients also reacted with NC2/COL. We conclude that NC2 and its adjacent collagenous segment mediate antiparallel dimer formation of collagen VII. Epidermolysis bullosa acquisita autoantibodies bound to this domain may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence.

Amino Acid Substitution↗
PubMed2001

Activation-dependent surface expression of LOX-1 in human platelets.

Lectin-like oxidized LDL receptor-1 (LOX-1) was initially identified as an oxidized LDL receptor in aortic endothelial cells. Here we identified LOX-1 mRNA and protein in human platelets in addition to recent findings on the expression in macrophages and smooth muscle cells. The presence of LOX-1 was further confirmed in the megakaryocytic cell lines. Flow cytometric analyses revealed that LOX-1 was exposed on the surface of platelets in an activation-dependent manner. Consistently, the activation-dependent binding of OxLDL to platelets was mostly inhibited by anti-LOX-1 antibody. Immunohistochemistry of the atherosclerotic plaque from a patient with unstable angina pectoris (UAP) revealed accumulation of LOX-1 protein at the site of thrombus. As LOX-1 recognizes and binds activated platelets, exposure of LOX-1 on activated platelets surface might assist thrombosis formation.

Animals↗
PubMed2001

The chemical nature of surface point defects on MoO3(010): adsorption of hydrogen and methyl.

We report density functional theory calculations using the Adaptive Coordinate Real-space Electronic Structure (ACRES) method of the terminal oxygen vacancy on the (010) surface of MoO3, within a (2 x 2) ordered array of vacancies on the surface. Analysis of the electronic structure of this surface shows that there are unoccupied dangling d(xz) and d(z)2 orbitals perpendicular to the surface that are created by the removal of terminal oxygen. The Mo-oxygen bonds surrounding the vacancy contract; however, the overall morphology of the surface is not drastically distorted. The vacancies alter the chemical character of the surface, as shown by studies of hydrogen and methyl binding. On both the "perfect" and vacancy surfaces, hydrogen was most strongly adsorbed over the terminal oxygen and most weakly bound over the symmetric bridging oxygen. Hydrogen is bound over the Mo atom, with a slightly smaller binding energy than hydrogen over the asymmetric bridging oxygen. The most favorable binding site for methyl on the vacancy surface is over the Mo atom exposed by removal of a terminal oxygen, whereas methyl bound to terminal oxygen is most stable on the perfect surface. There is no local minimum for adsorption over the symmetric bridging oxygen; instead, a methyl placed over this site moves toward the terminal oxygen vacancy. Analysis of the bonding shows that methyl is bound more strongly than hydrogen over the Mo atom because the C 2p orbital has better overlap with the Mo d(z)2 orbital than the hydrogen 1s. In addition, the steric repulsion observed for methyl over the perfect MoO3(010) surface is more easily relieved with the presence of the terminal oxygen vacancy.

Journal Article↗
PubMed2001

Effects of a lycopene-rich diet on spontaneous and benzo[a]pyrene-induced mutagenesis in prostate, colon and lungs of the lacZ mouse.

Consumption of lycopene has been associated with reduced risk of prostate cancer. We have investigated the effects of lycopene, fed as a lycopene-rich tomato oleoresin (LTO) at two doses, on in vivo mutagenesis in prostate, colon, and lungs of lacZ mice. Both short-term benzo[a]pyrene (BaP)- induced and long-term spontaneous mutagenesis were monitored. Non-significant inhibition of spontaneous mutagenesis in prostate and colon was observed at the higher dose of LTO, and the observation of inhibition in colon was facilitated by an unusually high spontaneous mutagenesis rate. BaP-induced mutagenesis was slightly inhibited by LTO in prostate. However, enhancement of BaP-induced-mutagenesis was observed in colon and lung. These results indicate that any antimutagenic effects of LTO may be organospecific.

Animals↗
PubMed2001

Where do Alzheimer's plaques and tangles come from? Aging-induced protein degradation inefficiency.

Amyloid plaques and neurofibrillary tangles are prominent lesions in the aging brain and they may be responsible for cell death in Alzheimer's disease. But a basic question has not been answered: why and how are plaques and tangles formed during aging? In this study, we approach this question by first examining what happens in the aging body. Plaques and tangles do not come alone, but together with many other aging markers in the body (cholesterol deposition, gallstones, hair graying, and bone loss, etc.). Because these aging markers occur to a certain extent in all elderly and at about the same time in life, it is reasonable to conceive that they originate from a common cause, that is, aging-induced metabolic inefficiency. If cholesterol and gallstone depositions are the results of inefficient degradation/clearance of lipids and minerals, then similarly plaque and tangle formation in most people would be the results of inefficient normal degradation of ?-amyloid precursor protein (APP) and tau, respectively. By this view, our studies should focus on the enzymes responsible for APP and tau normal degradation and their natural changes in aging, rather than on presumed pathological factors. Whatever precise mechanisms underlying their depositions, plaques and tangles are the natural products of aging, thus fundamentally different from pathological events such as cancer growth in concept.

Aging↗