Microchimerism and allograft acceptance: is it caused by microchimerism or graft antigens?
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Animals↗
Biomedical subjects
Y Hua
Publications and source records attributed to Y Hua.
Cytokine requirement for the development of rat dendritic cells by in vitro culturing of bone marrow cells.
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Animals↗
GM-CSF-independent development of dendritic cells from bone marrow cells in the GM-CSF-receptor-deficient mouse.
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Animals↗
Constituents of red yeast rice, a traditional Chinese food and medicine.
Detailed analyses were undertaken of the natural constituents of red yeast rice, a traditional Chinese medicine and food known for centuries to improve blood circulation. Preparation of red yeast rice following ancient methods by fermenting the fungal strain Monascus purpureus Went on moist and sterile rice indicated the presence of a group of metabolites belonging to the monacolin family of polyketides, together with fatty acids, and trace elements. The presence of these compounds may explain in part the cholesterol-lowering ability associated with this traditional Chinese food.
Anticholesteremic Agents↗
A metabolite of equine estrogens, 4-hydroxyequilenin, induces DNA damage and apoptosis in breast cancer cell lines.
Estrogen replacement therapy has been correlated with an increased risk of developing breast or endometrial cancer. 4-Hydroxyequilenin (4-OHEN) is a catechol metabolite of equilenin which is a minor component of the estrogen replacement formulation marketed under the name of Premarin (Wyeth-Ayerst). Previously, we showed that 4-OHEN autoxidizes to quinoids which can consume reducing equivalents and molecular oxygen, are potent cytotoxins, and cause a variety of damage to DNA, including formation of bulky stable adducts, apurinic sites, and oxidation of the phosphate-sugar backbone and purine/pyrimidine bases [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. All of these deleterious effects could contribute to the cytotoxic and genotoxic effects of equilenin in vivo. In the study presented here, we examined the relative toxicity of 4-OHEN in estrogen receptor (ER) positive cells (MCF-7 and S30) compared to that in breast cancer cells without the estrogen receptor (MDA-MB-231). The data showed that 4-OHEN was 4-fold more toxic to MCF-7 cells (LC(50) = 6.0 +/- 0. 2 microM) and 6-fold more toxic to S30 cells (LC(50) = 4.0 +/- 0.1 microM) than to MDA-MB-231 cells (LC(50) = 24 +/- 0.3 microM). Using the single-cell gel electrophoresis assay (comet assay) to assess DNA damage, we found that 4-OHEN causes concentration-dependent DNA single-strand cleavage in all three cell lines, and this effect could be enhanced by agents which catalyze redox cycling (NADH) or deplete cellular GSH (diethyl maleate). In addition, the ER(+) cell lines (MCF-7 and S30) were considerably more sensitive to induction of DNA damage by 4-OHEN than the ER(-) cells (MDA-MB-231). 4-OHEN also caused a concentration-dependent increase in the amount of mutagenic lesion 8-oxo-dG in the S30 cells as determined by LC/MS-MS. Cell morphology assays showed that 4-OHEN induces apoptosis in these cell lines. As observed with the toxicity assay and the comet assay, the ER(+) cells were more sensitive to induction of apoptosis by 4-OHEN than MDA-MB-231 cells. Finally, the endogenous catechol estrogen metabolite 4-hydroxyestrone (4-OHE) was considerably less effective at inducing DNA damage and apoptosis in breast cancer cell lines than 4-OHEN. Our data suggest that the cytotoxic effects of 4-OHEN may be related to its ability to induce DNA damage and apoptosis in hormone sensitive cells in vivo, and these effects may be potentiated by the estrogen receptor.
Apoptosis↗
Complement activation in the brain after experimental intracerebral hemorrhage.
OBJECT: Brain edema formation following intracerebral hemorrhage (ICH) appears to be partly related to erythrocyte lysis and hemoglobin release. Erythrocyte lysis may be mediated by the complement cascade, which then triggers parenchymal injury. In this study the authors examine whether the complement cascade is activated after ICH and whether inhibition of complement attenuates brain edema around the hematoma. METHODS: This study was divided into three parts. In the first part, 100 microl of autologous blood was infused into the rats' right basal ganglia, and the animals were killed at 24 and 72 hours after intracerebral infusion. Their brains were tested for complement factors C9, C3d, and clusterin (a naturally occurring complement inhibitor) by using immunohistochemical analysis. In the second part of the study, the rats were killed at 24 or 72 hours after injection of 100 microl of blood. The C9 and clusterin proteins were quantitated using Western blot analysis. In the third part, the rats received either 100 microl of blood or 100 microl of blood plus 10 microg of N-acetylheparin (a complement activation inhibitor). Then they were killed 24 or 72 hours later for measurement of brain water and ion contents. It was demonstrated on Western blot analysis that there had been a sixfold increase in C9 around the hematoma 24 hours after the infusion of 100 microl of autologous blood. Marked perihematomal C9 immunoreactivity was detected at 72 hours. Clusterin also increased after ICH and was expressed in neurons 72 hours later. The addition of N-acetylheparin significantly reduced brain edema formation in the ipsilateral basal ganglia at 24 hours (78.5 +/- 0.5% compared with 81.6 +/- 0.8% in control animals, p < 0.001) and at 72 hours (80.9 +/- 2.2% compared with 83.6 +/- 0.9% in control animals, p < 0.05) after ICH. CONCLUSIONS: It was found that ICH causes complement activation in the brain. Activation of complement and the formation of membrane attack complex contributes to brain edema formation after ICH. Blocking the complement cascade could be an important step in the therapy for ICH.
Animals↗
Tin-mesoporphyrin, a potent heme oxygenase inhibitor, for treatment of intracerebral hemorrhage: in vivo and in vitro studies.
Spontaneous intracerebral hemorrhage (ICH) is the stroke subtype with highest mortality and morbidity. ICH can also occur following traumatic brain injury and thrombolysis for ischemic stroke and myocardial infarction. Development of ICH-induced hemispheric edema can elevate intracranial pressure and cause death. In survivors, edema-related white matter injury can lead to life-long neurological deficits. At present, there are no scientifically proven treatments for ICH. Heme oxygenase products, particularly iron and bilirubin, can be toxic to cells. In cerebral ischemia models, metalloporphyrins that are potent heme oxygenase inhibitors, reduce edema and infarct size. Tin-mesoporphyrin (SnMP) is a neuroprotectant that has also been used clinically to treat hyperbilirubinemia. Presently, we tested the hypothesis that SnMP treatment would reduce edema development following experimental ICH. We produced hematomas in pentobarbital-anesthetized pigs (9-11 kg) by infusing autologous blood into the frontal white matter. To maximize tissue concentrations, SnMP (87.5 microM in DMSO) or DMSO (vehicle controls) was included in the infused blood. Pig brains were frozen in situ at 24 hrs. following ICH and hematoma and edema volumes were determined on coronal sections by computer-assisted image analysis. We also examined the effects of SnMP in vitro on ferritin iron release, the formation of iron-induced thiobarbituric acid reactive substances (TBARS) and initial clot formation and hemolysis. SnMP treatment significantly reduced intracerebral mass following ICH. This was due to significant decreases in hematoma (0.68+/-0.08 vs. 1.39+/-0.30 cc, vehicle controls p<0.025) and edema volumes (edema = 1. 16+/-0.33 vs. 1.77+/-0.31 cc, p<0.05). In vitro, SnMP did not stabilize ferritin iron against reductive release nor did it decrease iron-induced TBARS formation in brain homogenates. SnMP or DMSO added to pig blood did not alter clot weights. In conclusion, SnMP reduced intracerebral mass in an ICH model by decreasing both hematoma and edema volumes SnMP's mechanism of action is presently unknown but may involve its potent inhibition of heme oxygenase activity. SnMP's effect appears unrelated to ferritin iron release, antioxidant activity or initial clot formation. Since SnMP treatment could be brain protective following ICH, further investigations into neurological and neuropathological outcomes and as well as into its mechanism of action are warranted.
Animals↗
Local interactions drive the formation of nonnative structure in the denatured state of human alpha-lactalbumin: a high resolution structural characterization of a peptide model in aqueous solution.
There are a small number of peptides derived from proteins that have a propensity to adopt structure in aqueous solution which is similar to the structure they possess in the parent protein. There are far fewer examples of protein fragments which adopt stable nonnative structures in isolation. Understanding how nonnative interactions are involved in protein folding is crucial to our understanding of the topic. Here we show that a small, 11 amino acid peptide corresponding to residues 101-111 of the protein alpha-lactalbumin is remarkably structured in isolation in aqueous solution. The peptide has been characterized by 1H NMR, and 170 ROE-derived constraints were used to calculate a structure. The calculations yielded a single, high-resolution structure for residues 101-107 that is nonnative in both the backbone and side-chain conformations. In the pH 6.5 crystal structure, residues 101-105 are in an irregular turn-like conformation and residues 106-111 form an alpha-helix. In the pH 4.2 crystal structure, residues 101-105 form an alpha-helix, and residues 106-111 form a loopike structure. Both of these structures are significantly different from the conformation adopted by our peptide. The structure in the peptide model is primarily the result of local side-chain interactions that force the backbone to adopt a nonnative 310/turn-like structure in residues 103-106. The structure in aqueous solution was compared to the structure in 30% trifluoroethanol (TFE), and clear differences were observed. In particular, one of the side-chain interactions, a hydrophobic cluster involving residues 101-105, is different in the two solvents and residues 107-111 are considerably more ordered in 30% TFE. The implications of the nonnative structure for the folding of alpha-lactalbumin is discussed.
Circular Dichroism↗
Solution reactivity of brevetoxins as monitored by electrospray ionization mass spectrometry and implications for detoxification.
The reactivities of brevetoxin compounds in acid and base and under oxidizing conditions were studied using electrospray ionization mass spectrometry (ESI-MS) to monitor reaction products. Brevetoxins are determined to be unstable in acidic and basic solutions. Under acidic conditions, brevetoxins containing an aldehyde functional group in the terminal "tail" side chain are easily converted to acetal structures, while "head" side lactone ring opening proceeds more slowly. Measurement of reaction rate constants indicates the following order of reactivity under acidic conditions: Btx-1 > Btx-2 > Btx-9. Under basic conditions, hydroxide ion attack at the head portion leads to lactone ring opening. Base hydrolysis (0.01 N NaOH in 50:50 methanol/water) goes to completion in 120 min for Btx-2 and Btx-9, but Btx-1 did not react to completion. Both acid and base hydrolyses can lead to reversible lactone ring opening, but base hydrolysis proceeds faster than acid hydrolysis under comparable conditions. Acid treatment is not an effective method for detoxifying brevetoxins. Base treatment can open the lactone ring (type B brevetoxins proceed faster than type A brevetoxins), leading to a product that is reportedly nontoxic, but the reaction is reversible. Brevetoxins are shown to be readily oxidized by permanganate in an irreversible and relatively fast reaction, likely through addition to double bonds followed by bond cleavage, suggesting that it is a viable method for detoxification.
Animals↗
Attenuation of thrombin-induced brain edema by cerebral thrombin preconditioning.
BACKGROUND AND PURPOSE: Edema formation after intracerebral hemorrhage has been linked to thrombin toxicity induced by the clot. However, thrombin at low concentrations actually protects neurons and astrocytes in culture from hypoglycemic and ischemic cell death. It is also known that a brief episode of brain ischemia increases neuronal tolerance to a subsequent severe ischemic episode. The objective of this study was to investigate whether pretreatment of the brain with low-dose thrombin induces tolerance to a subsequent large dose of thrombin injected into brain parenchyma. METHODS: The rat brain was preconditioned with 1 U thrombin by direct infusion into the right caudate nucleus. After thrombin pretreatment, the effects of a large dose (5 U) of thrombin on brain edema formation were studied at different intervals. We examined whether heat-shock protein (HSP) 27, HSP32, and HSP70 were induced by Western blot analysis, immunocytochemistry, and immunofluorescent double staining. RESULTS: Thrombin pretreatment significantly attenuated the brain edema that normally follows the infusion of a large dose of thrombin (79.2+/-0.4 versus 84.0+/-0.3; P<0.01). This effect was abolished by the thrombin inhibitor hirudin. Time course studies showed that the maximal effect of thrombin preconditioning (TPC) on brain edema formation was 7 days after pretreatment. This time course corresponded to marked upregulation of HSP27 in the ipsilateral brain. TPC also induced HSP32, but this effect occurred earlier than the effect on edema formation. TPC had no effect on HSP70. Immunocytochemistry and immunofluorescent double labeling showed that HSP27 and HSP32 were expressed in astrocytes after TPC. CONCLUSIONS: OFF phenomenon of thrombin-induced tolerance of the brain to edema formation may be related to HSP27 induction.
Animals↗
Quantitation of sheep-associated malignant catarrhal fever viral DNA by competitive polymerase chain reaction.
A single-step, competitive polymerase chain reaction technique was developed to quantitate sheep-associated malignant catarrhal fever (SA-MCF) viral DNA. The assay employed coamplification of a fixed quantity of target DNA with graded amounts of a competitor, generated by truncation of the target sequence lying between the 2 primer binding sites. The assay yielded a linear response (r = 0.98) for DNA measurement within the range of 30-300,000 copies. Amplification efficiency analysis by coamplification of target and competitor in equal copy numbers for various numbers of cycles showed that the relative abundance of the coamplified products remained constant with increasing cycle numbers up to 40. Reproducibility was assessed by repetitively assaying a set of blind-coded samples from a variety of animals and tissues. Results indicated that the assay is reliable and reproducible for quantitation of SA-MCF viral DNA in samples from asymptomatically infected sheep and from animals with clinical SA-MCF.
Animals↗
Ultra-early clot aspiration after lysis with tissue plasminogen activator in a porcine model of intracerebral hemorrhage: edema reduction and blood-brain barrier protection.
OBJECT: Ultra-early hematoma evacuation (< 4 hours) after intracerebral hemorrhage (ICH) may reduce mass effect and edema development and improve outcome. To test this hypothesis, the authors induced lobar hematomas in pigs. METHODS: The authors infused 2.5 ml of blood into the frontal cerebral white matter in pigs weighing 8 to 10 kg. In the treatment group, clots were lysed with tissue plasminogen activator ([tPA], 0.3 mg) and aspirated at 3.5 hours after hematoma induction. Brains were frozen in situ at 24 hours post-ICH and hematomal and perihematomal edema volumes were determined on coronal sections by using computer-assisted morphometry. Hematoma evacuation rapidly reduced elevated cerebral tissue pressure from 12.2+/-1.3 to 2.8+/-0.8 mm Hg. At 24 hours, prior clot removal markedly reduced hematoma volumes (0.40+/-0.10 compared with 1.26+/-0.13 cm3, p < 0.005) and perihematomal edema volumes (0.28+/-0.05 compared with 1.46+/-0.24 cm3, p < 0.005), compared with unevacuated control lesions. Furthermore, no Evans blue dye staining of perihematomal edematous white matter was present in brains in which the hematomas had been evacuated, compared with untreated controls. CONCLUSIONS: Hematomas were quickly and easily aspirated after treatment with tPA, resulting in significant reductions in mass effect. Hematoma aspiration after fibrinolysis with tPA enabled removal of the bulk of the hematoma (> 70%), markedly reduced perihematomal edema, and prevented the development of vasogenic edema. These findings in a large-animal model of ICH provide support for clinical trials that include the use of fibrinolytic agents and ultra-early stereotactically guided clot aspiration for treating ICH.
Animals↗
[Changes of serum elastase, alpha 1-antitrypsin, procollagen III peptide and malonaldehyde in smokers with and without chronic obstructive pulmonary diseases].
OBJECTIVE: Serum elastase, alpha(1)-antitrypsin (alpha(1)-AT), procollagen III peptide (PIIIP) and malonaldehyde (MDA) were studied in smokers with and without chronic obstructive pulmonary disease (COPD) to explore the roles of these factors in COPD induced by smoking. METHODS: A design of 154-pair case-control study was used. alpha(1)-AT, MDA and PIIIP were determined with colorimetric method, fluo-spectrophotometric method and radio-immunological assay, respectively, in non-smoker control and smokers with and without COPD. RESULTS: Serum PIIIP content was increased in smokers and alpha(1)-AT decreased significantly in patients with COPD. CONCLUSION: Decreased alpha(1)-AT may be a susceptible factor in the development of COPD caused by smoking.
Adult↗
[Dose-related response of adenosine in hypoxic pulmonary hypertension in canines].
The vasodilator test is of critical importance in determining the reversibility of pulmonary hypertension and hence the indication of surgical treatments for cases of marginal pulmonary hypertension associated with left to right shunt. This study aimed at the dose-related vasodilation effect of adenosine (AD) on hypoxic pulmonary hypertension. The hemodynamic indexes were examined in 8 canines with acute hypoxic pulmonary hypertension after administration of intravenous adenosine. The results showed that AD decreased both systemic and pulmonary pressures in a dose-dependent way, with very close linear correlation; so did the systemic and pulmonary resistance (r = -0.970 and -0.994, respectively; P < 0.001). The PASP reductions were significantly greater than SASP reductions at AD doses of 100 to 200 micrograms/(kg.min) (from -11.0% +/- 4.2% to -27.7% +/- 14.8% vs from -2.6% +/- 7.4% to -14.9% +/- 7.0%, respectively), while PVR reductions were significantly greater than SVR reductions (-18.8% +/- 8.05% vs -10.2% +/- 6.86% and -29.8% +/- 14.46% vs -21.1% +/- 9.53%, respectively, P < 0.05). AD increased cardiac output and cardiac index to the significant degree only when the dosage increased to 300 micrograms/(kg.min) or above. These results suggest that AD seems to be the choice of drug in vasodilator test of left to right shunt pulmonary hypertension, and the proper dosage ranges from 100 to 200 micrograms/(kg.min).
Adenosine↗
[Influence of intravenous-immunoglobulin on T lymphocyte subsets, NK cell and some cytokines in mice with experimental viral myocarditis].
This study sought to gain an insight into the immunological pathogenesis of viral myocarditis and the mechanism of therapeutic action of intravenous-immunoglobulin (IVIG) on the disease, BALB/c mice were randomized into four groups: normal control group; "myocarditis model group", inoculated intraperitoneally with CVB3; "IVIG protection group", injected intraperitoneally with CVB3 and IVIG; and "IVIG treatment groups", injected intraperitoneally with CVB3 and IVIG. The amount of T cell in different T cell subsets and the activities of NK cell, IL-1 and IL-2 were assayed using spleen specimen while peripheral blood was used to measure TNF activity. The results showed: that in comparison with normal control, all spleen T cell subsets decreased in amount in myocarditis model group, so did its NK cell activity while its IL-1, IL-2 and TNF activities increased significantly. When compared with myocarditis model group, however, both IVIG protection group and IVIG treatment group showed significant elevation of NK cell activity and T cell subsets but reduced IL-1, IL-2 and TNF activities with no remarkable change in T cell subsets. The results of suggest that the immunological injury mediated by T cells plays a critical role in the pathogenesis of viral myocarditis. The protective and therapeutic effects of IVIG on the murine CVB3 myocarditis and the related immunological evidence of its actions may indicate the prospect that IVIG will become a potent and safe treatment for viral myocarditis in human.
Animals↗
Conformational analysis of the interdomain linker of the central homology region of chloroplast initiation factor IF3 supports a structural model of two compact domains connected by a flexible tether.
A peptide corresponding to the interdomain linker of chloroplast IF3 has been synthesized and its structure studied by NMR and CD as a function of temperature and pH. At low temperature and neutral pH the apparent helical content is 25%. pH and ionic strength dependent CD studies demonstrate that sidechain-sidechain interactions stabilize the structure observed at low temperature. The helicity decreases with temperature and above 25 degrees C the peptide is less than 15% helical. These results indicate that the peptide has little intrinsic tendency to form helical structure at physiologically relevant temperatures and strongly suggests that the linker region is flexible in intact chloroplast IF3.
Amino Acid Sequence↗
Characterization of the mouse neurofilament light (NF-L) gene promoter by in vitro transcription.
We have used in vitro transcription to access the basic sequences and factors required for the transcription of the mouse neurofilament light promoter (pNF-L) in the absence of chromatin structure. Deletion from -1.7 to -154 results in little change in NF-L promoter activity using nuclear extracts from either brain (expressing) or liver (non-expressing) tissues. Further deletion to -29 results in a gradual five-fold drop in promoter activity in both extracts. Only replacement of the entire -148 to -29 region results in a drop in NF-L promoter activity to basal levels. Thus, the NF-L promoter differs from the mouse NF heavy (NF-H) and mid-sized (NF-M) promoters in that no specific sequence within the immediate upstream NF-L promoter region (-154 to -29) appears to be responsible for enhancement or brain-specific transcription. We show that the order of strength of the three NF promoters is NF-H>NF-M>NF-L and identify sequences that can increase or reduce transcription when placed in front of heterologous NF promoters. We conclude that the NF-L promoter is a modular, weak and promiscuous promoter whose regulation differs from NF-H or NF-M. Our data suggest that chromatin structure may play an important role in the regulation of the NF-L promoter.
Animals↗
On the global architecture of initiation factor IF3: a comparative study of the linker regions from the Escherichia coli protein and the Bacillus stearothermophilus protein.
Initiation factor IF3 is a protein involved in the initiation stage of protein synthesis. It consists of two global domains linked by a 20 residue long, solvent-exposed linker. Recently, the structure of the N and C-terminal domains of the Bacillus stearothermophilus protein have been solved by X-ray crystallography and the structure of the intact Escherichia coli protein has been studied by NMR. These two studies have led to apparently contradictory models for the domain organization of IF3. The NMR study of the E. coli protein indicates that the linker region is flexible, while the studies of the isolated N and C-terminal domains of the B. stearothermophilus protein suggest that the linker forms a rigid helical rod. In order to resolve this discrepancy, a set of peptides corresponding to the linker regions of the B. stearothermophilus and the E. coli protein were synthesized. Circular dichroism and NMR spectroscopy were used to study the helical content as a function of pH, temperature, peptide concentration and ionic strength. Both peptides are monomeric. The estimated helical content of the linker fragment from B. stearothermophilus is 68% at high pH and 1 degree C. The measured helicity decreases to 53% at pH 7.0 and 1 degree C. In contrast, the peptide corresponding to the E. coli IF3 linker region is largely unstructured with a maximum helical content of 15% at high pH and only 8% at pH 7.0, 1 degree C. These results suggest that the different structures observed for the two intact proteins may be due to the different intrinsic stability of the two linker peptides. The helical content of the two linker peptides is, however, much closer when the peptides are compared at the respective temperatures of optimum growth for E. coli and B. stearothermophilus (3% versus 17%). The pH and ionic strength dependence of the helical content of the B. stearothermophilus peptide demonstrates that side-chain/side-chain interactions play an important role in stabilizing the helical structure. In addition, studies with mutant peptides show that the first Asp residue in the linker sequence helps to stabilize the helix via an N- capping interaction.
Amino Acid Sequence↗