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Biomedical subjects

Q Zhao

Publications and source records attributed to Q Zhao.

At least 217 records · Page 12Linked to original sources

Peptic hemoglobin hydrolysis in an ultrafiltration reactor at pilot plant scale generates opioid peptides.

Two hemorphins, peptides with opioid activity, have been isolated from a pepsin hydrolysate of bovine hemoglobin, by use of gel permeation (GP) and reverse phase (RP) high-performance liquid chromatography (HPLC). Their primary structure and accurate molecular weights, determined by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, were identical to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin 7) of the beta-chain of bovine hemoglobin. Two other peptides, 34-40 (hemorphin-7) and 34-41 (hemorphin-8) of the beta-chain of bovine hemoglobin, have been synthesized and studied. The opioid potency of these peptides, exhibited by the use of electrically stimulated muscle of isolated guinea pig ileum (GPI), were significant and comparable with some others previously described. Studies of opioid activities and primary structure of hemorphins led us to postulate the important role of arginine and phenylalanine in opioid potency.

Amino Acid Sequence↗

Hyperthermia nullifies the ameliorating effect of dizocilpine maleate (MK-801) in focal cerebral ischemia.

The present study was inspired by two previous findings from the laboratory. The first was that dizocilpine maleate (MK-801) fails to reduce infarct size when the middle cerebral artery (MCA) is permanently occluded by an intraluminal filament technique in rats. In seeking the reasons for this we measured temperature and found that the body temperature of occluded animals increases to 39.0-39.5 degrees C during the first 2-3 h. In order to explore whether the rise in temperature was responsible for the lack of effect of MK-801, two groups of animals were studied, both containing animals which were subjected to 2 h of transient MCA occlusion and given MK-801 15 min before, as well as 6 and 24 h after ischemia. In one group, temperature was allowed to rise spontaneously during ischemia (39.0-39.5 degrees C). In the other, body temperature was maintained close to normal during ischemia, and for the first 6 h postischemically, by cooling of the ambient air. Infarct volume was assessed by triphenyltetrazolium chloride staining after 48 h of recovery. The results showed that MK-801 failed to reduce infarct size in animals whose body temperature rose during ischemia. In contrast, the drug markedly reduced infarct volume in temperature-controlled animals; in fact, 5/8 animals had no infarcts but selective neuronal damage only. The results suggest that amelioration of focal ischemic damage cannot be expected if body and brain temperature is allowed to rise above normal.

Animals↗

Enzymatic and nonenzymatic polarizations of alpha,beta-unsaturated ketosteroids and phenolic steroids. Implications for the roles of hydrogen bonding in the catalytic mechanism of delta 5-3-ketosteroid isomerase.

Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17 beta-estradiol and 17 beta-dihydroequilenin), which are potent competitive inhibitors of delta 5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme-steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17 beta-estradiol and 17 beta-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a lambda max of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a lambda max of 258.5 nm in aqueous solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗

Glass wool-H2O2/CoCl2 test system for in vitro evaluation of biodegradative stress cracking in polyurethane elastomers.

Environmental stress cracking (ESC) in polyetherurethanes has been demonstrated in animal models. However, duplication of this phenomenon in vitro has been a challenge. The glass wool-H2O2/CoCl2 test system was designed to provide oxidation components found in vivo, i.e., hydroxyl radical (HO.), molecular oxygen (O2), and superoxide (O2-.) required for auto-oxidation of polyetherurethanes. The in vitro test is not only reliable in duplicating the characteristics of in vivo stress cracking in polyetherurethanes at 37 degrees C, but also accelerates ESC as much as seven times. The test results on several types of polyurethanes showed that Tecothane 80A was as vulnerable to biodegradative stress cracking in vitro as Pellethane 2363-80A, while the new generation of polyurethanes, Corethane, demonstrated great resistance against stress cracking.

Biodegradation, Environmental↗

Degradation potential of plasma-sprayed hydroxyapatite-coated titanium implants.

The purpose of this study was to develop an analytical method for evaluating the dissolution behavior of plasma-sprayed hydroxyapatite (HAP) coatings. Six commercially available and clinically used coatings applied to nonporous titanium coupons according to their respective specifications for orthopedic devices were used in this study. Dissolution behavior was monitored by first preconditioning the implant in 0.15 mol L-1 sodium chloride solution at 37 degrees C until either equilibrium or the desired change in solution hydrogen and calcium ion concentrations had been reached. In the second step, the implants were subjected to dual constant composition (DCC) dissolution under conditions of controlled undersaturation. Results indicate that the dissolution rates of the HAP coatings may differ by as much as a factor of 5 despite the fact that analytical techniques, including X-ray diffraction and FTIR, indicate no change in crystallinity or composition of the coatings before and after the solution treatment. These results indicate that HAP coatings from different sources react very differently when placed in the same local aqueous environment. However, more work will be necessary before these in vitro results can be used to predict in vivo behavior.

Alloys↗

Regulation of brain-derived neurotrophic factor gene expression after transient middle cerebral artery occlusion with and without brain damage.

Levels of mRNA for c-fos, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), TrkB, and TrkC were studied using in situ hybridization in the rat brain at different reperfusion times after unilateral middle cerebral artery occlusion (MCAO). Short-term (15 min) MCAO, which does not cause neuronal death, induced elevated BDNF mRNA expression confined to ipsilateral frontal and cingulate cortices outside the ischemic area. With a longer duration of MCAO (2 h), which leads to cortical infarction, the increase was more marked and elevated BDNF mRNA levels were also detected bilaterally in dentate granule cells and CA1 and CA3 pyramidal neurons. Maximum expression was found after 2 h of reperfusion. At 24 h BDNF mRNA expression had returned to control values. In the ischemic core of the parietal cortex only scattered neurons were expressing high levels of BDNF mRNA after 15 min and 2 h of MCAO. Analysis of different BDNF transcripts showed that MCAO induced a marked increase of exon III mRNA but only small increases of exon I and II mRNAs in cortex and hippocampus. In contrast to BDNF mRNA, elevated expression of c-fos mRNA was observed in the entire ipsilateral cerebral cortex, including the ischemic core, after both 15 min and 2 h of MCAO. Two hours of MCAO also induced transient, bilateral increases of NGF and TrkB mRNA levels and a decrease of NT-3 mRNA expression, confined to dentate granule cells. The upregulation of BDNF mRNA expression in cortical neurons after MCAO is probably triggered by glutamate through a spreading depression-like mechanism. The lack of response of the BDNF gene in the ischemic core may be due to suppression of signal transduction or transcription factor synthesis caused by the ischemia. The observed pattern of gene expression after MCAO agrees well with a neuroprotective role of BDNF in cortical neurons. However, elevated levels of NGF and BDNF protein could also increase synaptic efficacy in the postischemic phase, which may promote epileptogenesis.

Animals↗

Glutamate, calcium, and free radicals as mediators of ischemic brain damage.

Calcium is considered a mediator of ischemic brain damage whether this is due to global or forebrain ischemia or to focal ischemia. Supporting evidence is the translocation of extracellular calcium into cells during ischemia, the precipitous rise in the free cytosolic calcium concentration, and the role of calcium in activating lipases, proteases, kinases, phosphatases, and endonucleases in potentially harmful metabolic cascades. In vitro and in vivo experiments suggest that the main route of entry is through channels gated by glutamate receptors. These experiments led to the excitotoxic hypothesis of cell death. The in vitro experiments further support the role of calcium as a mediator of cell death. Both cell calcium overload and acidosis enhance the production of partially reduced oxygen species, thus predisposing to free radical-related damage. In transient global or forebrain ischemia, free radicals formed during reperfusion may contribute to a perturbed membrane function, leading to a sustained alteration of cell calcium metabolism with ultimate mitochondrial calcium overload. In focal ischemia (stroke), free radicals may be important mediators of the infarction process. Infarction can be regarded as a form of secondary damage, which is probably caused by microvascular dysfunction. Very likely, such dysfunction is triggered by upregulation of adhesion molecules such as ICAM-1, microvascular "plugging," and an inflammatory response at the blood-endothelial cell interface. The involvement of free radicals in this type of secondary damage is supported by results showing that nitrones that act as free radical spin-traps ameliorate focal ischemic damage with a therapeutic window of many hours.

Animals↗

VV-hemorphin-7 and LVV-hemorphin-7 released during in vitro peptic hemoglobin hydrolysis are morphinomimetic peptides.

Two opioid peptides were generated by in vitro pepsin treatment of bovine hemoglobin. These peptides were identified using a GPI test and purified using HPLC chromatographic techniques. They correspond to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin-7) of the beta-chain of bovine hemoglobin. Binding experiments strongly confirm that VV-hemorphin-7 and LVV-hemorphin-7 are opioid peptides since they inhibited [3H]naloxone binding to rat brain membranes. Our results indicate that VV-hemorphin-7 and LVV-hemorphin-7 exhibit a lesser potency both in GPI and binding tests. Selectivity and affinity of these purified peptides and synthetic hemorphin-7 for opioid receptors is discussed.

Animals↗

Physiological characterization of a cadmium-resistant mutant in the fission yeast Schizosaccharomyces pombe.

After treatment of the wild-type strain 975 h+ with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutants able to grow on YEP-medium containing 15 mM CdCl2 were isolated. Mutant R17 showed a specific resistance to Cd, but no increased tolerance to Pb(NO3)2, CuSO4 or ZnCl2. No higher content of Cd-binding phytochelatins could be detected, but the mutant accumulated lower levels of Cd than the corresponding wild-type. No change, however, was observed in the accumulation of Cu. Under the influence of Cd, the respiratory activity of mutant R 17 is reduced less than in the wild-type. The mechanism of resistance could be an increased secretion or a decreased uptake of Cd.

Cadmium↗

Use of cyclodextrin and its derivatives as carriers for oligonucleotide delivery.

The use of antisense phosphorothioate oligodeoxynucleotides as tools for modulating gene expression represents a novel strategy for designing drugs to treat a variety of diseases. Several factors, including cellular uptake and internalization of the phosphorothioate oligodeoxynucleotide, are important parameters in determining the effectiveness of antisense agents as therapies. We have used cyclodextrin and its analogs as carriers to increase cellular uptake of phosphorothioate oligodeoxynucleotides. The studies were carried out using 35S-labeled and fluorescent-labeled phosphorothioate oligodeoxynucleotide in human T cell leukemia H9 cell line. Cellular uptake of phosphorothioate oligodeoxynucleotide in the presence of cyclodextrin was found to be concentration and time dependent. Using various cyclodextrin analogs, e.g., 2-hydroxypropyl beta-cyclodextrin (HPCD), hydroxyethyl beta-cyclodextrin (HECD), and a mixture of various hydroxypropyl beta-cyclodextrins (Encapsin), we observed increases in phosphorothioate oligodeoxynucleotide uptake, up to twofold to threefold in 48 hours. Confocal microscopy studies confirmed that oligonucleotide was present intracellularly. Cyclodextrin itself was not toxic at the concentration used. Cyclodextrins did not seem to affect the efflux of phosphorothioate oligodeoxynucleotide from cells. Stability of phosphorothioate oligodeoxynucleotide against endogenous cellular nucleases remained unchanged in the presence of cyclodextrins. These studies suggest that cyclodextrin and its analogs might be used successfully as carriers for oligonucleotide and analogs.

Amino Acid Sequence↗

Mechanisms of secondary brain damage in global and focal ischemia: a speculative synthesis.

The objective of this article is to amalgamate previous results into a speculative synthesis that sheds light on the causes of secondary brain damage following either global/forebrain or focal ischemia. The hypothesis is based on the well-founded assumption that the pathophysiology of the brain damage incurred by global or forebrain ischemia is different from that of focal ischemia. In the former, the ischemia is usually dense and of brief duration and, provided that reperfusion is adequate, cell damage is conspicuously delayed, mostly affecting selectively vulnerable neurons. In contrast, focal ischemia is either long-lasting or permanent, and it is usually less severe, particularly in the perifocal penumbral regions. The lesion is typically pan-necrotic ("infarction"), initially affecting the focus supplied by the occluded artery, later invading the penumbra zone. Available results allow a restatement of the calcium hypothesis of cell death. In global or forebrain ischemia, calcium influx through channels gated by voltage or glutamate receptors is envisaged to trigger reactions that limit the survival of neurons during reperfusion, leading to secondary neuronal death after hours or days of survival. It can be hypothesized that the initial insult leads to a sustained alteration of membrane calcium handling, resulting in slow, gradual calcium overload of mitochondria. Alternatively, a sustained perturbation of the intracellular signal transduction pathway leads to changes in transcription or translation, bereaving the cells of heat shock and stress proteins, of trophic factors, or of enzymes required for survival. However, with the possible exception of the gerbil, neither microvascular failure nor primary mitochondrial dysfunction is believed to be involved. In focal ischemia, similar reactions are probably triggered by calcium influx, whether this is sustained (the focus) or intermittent (the penumbra). However, these play a minor role in cell death since they are overridden by reactions producing mediators of rapidly developing secondary damage, affecting either microvessels or mitochondria. Very probably, some of these mediators are free radicals, or nitric oxide, or other reactive metabolites, emanating from lipid hydrolysis and arachidonic acid metabolism. During continuous ischemia, or during recirculation following 1-3 h of ischemia, these mediators activate adhesion molecules in endothelial cells or polymorphonuclear leucocytes, or oxidize key proteins. The result is either failure of microcirculation ("capillary plugging"), or sustained mitochondrial failure. Since calcium influx is an initial event, agents reducing presynaptic depolarization and calcium entry through glutamate receptor-gated and other calcium channels have predictably a narrow therapeutic window; however, since spin trapping agents of the nitrone class act many hours after the induction of focal ischemia, their therapeutic window is potentially very wide. This may be because expression of mRNAs for adhesion molecules and their synthesis are relatively slow processes, and because the nitrones act on events that involve adhesion of leukocytes to the endothelial cells, with plugging of capillaries and postcapillary venules, and on the ensuing inflammatory response.

Animals↗

Efficient excision of the upstream large intron from P4-generated pre-mRNA of the parvovirus minute virus of mice requires at least one donor and the 3' splice site of the small downstream intron.

We have previously shown that efficient excision of the upstream large intron from P4-generated pre-mRNA of the autonomous parvovirus minute virus of mice depends upon at least the initial presence of sequences within the downstream small intron (Q. Zhao, R. V. Schoborg, and D. J. Pintel, J. Virol. 68:2849-2859, 1994). In this report, we show that the requirement of downstream small intron sequences is complex and that efficient excision of the upstream intron requires at least one small intron donor and the 3' splice site. In the absence of both small intron donors, a new spliced product is produced in which the intervening exon is skipped and the large intron donor at nucleotide 514 is joined to a small intron acceptor. Exon skipping caused by the loss of the two small intron donors can be overcome, and the excision of the large intron can be regained by mutations that improve the large intron polypyrimidine tract. These results are consistent with a model in which the binding of multiple splicing factors that assemble at both a downstream donor and acceptor facilitates the binding of splicing factors to the weak polypyrimidine tract of the upstream large intron, thereby defining the intervening exon and promoting excision of the upstream intron.

Animals↗

Sequences within the parvovirus minute virus of mice NS2-specific exon are required for inclusion of this exon into spliced steady-state RNA.

When the minute virus of mice NS2-specific exon was modified by either substitution or deletion, most P4-generated pre-mRNA was spliced from the large-intron donor at nucleotide 514 to the small-intron acceptor at nucleotide 2377. Improvement to consensus of large-intron splice sites in such mutants did not suppress exon skipping or restore large-intron excision. Therefore, sequences within the NS2-specific exon are required for inclusion of this exon into spliced, steady-state minute virus of mice RNA.

Animals↗

Airway epithelial cell responses to ozone injury.

The airway epithelial cells is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products--hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines--macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretory leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease.

Animals↗

Activation of eicosanoid metabolism in human airway epithelial cells by ozonolysis products of membrane fatty acids.

Inhaled ozone can react with a variety of cellular macromolecules within the lung. Recent analyses of the chemistry of ozone reactions with unsaturated fatty acids, which are present in all membranes and in mucus in the airways, indicate that ozonolysis yields one aldehyde and one hydroxyhydroperoxide molecule for each molecule of ozone. The hydroxyhydroperoxide molecule is unstable in aqueous environments, and subsequently yields a second aldehyde and hydrogen peroxide. The structure of common unsaturated fatty acids is such that attack by ozone at the carbon-carbon double bonds will yield 3-, 6-, and 9-carbon saturated and unsaturated aldehydes and hydroxyhydroperoxide. This study examines the effects of ozonolysis products on eicosanoid metabolism in human airway epithelial cells. Eicosanoid biosynthesis is important in a wide array of pathophysiological responses in the airway, and the release of eicosanoids by the epithelial barrier is likely to be significant in diseases induced by environmental factors. Previously, we demonstrated that ozone can increase eicosanoid synthesis from airway epithelial cells exposed in vitro. Human exposures to concentrations of ozone below the current National Ambient Air Quality Standard (0.12 ppm, not to be exceeded for more than one hour once per year) also resulted in increased eicosanoids in bronchoalveolar lavage fluid. To determine whether ozonolysis products could activate eicosanoid release, we exposed human airway epithelial cells to 3-, 6-, and 9-carbon aldehydes, hydroxyhydroperoxides, and hydrogen peroxide. We measured (1) eicosanoid metabolism using high-performance liquid chromatography and radioimmunoassays, and (2) the effects of the aldehydes, hydroxyhydroperoxides, and hydrogen peroxide on cell lysis. Eicosanoid release increased after exposure to aldehyde; release induced by 9-carbon (nonanal) aldehyde was greater than that induced by the 6-carbon (hexanal) or 3-carbon (propanal) aldehydes. Hydroxyhydroperoxides induced greater eicosanoid release than the corresponding aldehydes of equivalent chain length. Again, the longer the aliphatic chain length of the hydroxyhydroperoxide the greater the effect. These effects were noted at concentrations of hydroxyhydroperoxide below those that produce cell lysis, and the time course of the two responses was dissimilar. Because hydroxyhydroperoxides can degrade into an aldehyde and hydrogen peroxide, it is conceivable that the effects observed were attributable to the formation of either hydrogen peroxide or hydrogen peroxide and aldehyde. This mechanism is unlikely, however, because the effects of hydroxyhydroperoxides on eicosanoid release were dependent on chain length, whereas each hydroxyhydroperoxide can produce only one hydrogen peroxide molecule. Although hydrogen peroxide alone also stimulated eicosanoid metabolism, this effect was not augmented when aldehyde and hydrogen peroxide were added together. In addition, the dose of hydroxyhydroperoxide needed to produce an effect (10 to 100 microM) was lower than that of hydrogen peroxide (300 microM). We could not fully evaluate the effects of the unsaturated aldehydes and hydroxyhydroperoxides. Although the 6-carbon and 9-carbon cis-3-aldehydes could be synthesized from the cis-3-alcohols, the resulting aldehydes were not chemically stable. The cis-3-aldehydes were useful for producing the corresponding 1-hydroxy-alkenyl-hydroperoxides of high purity. These results support the method selected for chemical synthesis, but further studies are required to establish proper storage and handling methods before these compounds can be tested in assays of eicosanoid metabolism.

Air Pollutants↗

[Modifications of gene expression by tumor promoters].

The modifications of gene expression by tumor promoters were analyzed in vitro and in vivo. The results of slot blot hybridizations showed that tumor promoter TPA induced c-fos and c-myc expressions in mouse fibroblast cell line BALB/3T3 and rat liver, decreased the levels of Rb RNA in BALB/3T3 cell line and of alpha 1-I3 RNA in rat liver. It was also demonstrated that tumor promoter phenobarbital influenced c-fos and c-myc expressions and decreased alpha 1I3 mRNA level in rat liver during a long term experiment. Phenobarbital was found to have no effect on c-fos and c-myc expressions in rat liver during a short experiment. Tumor promoters induced the expressions of c-fos and c-myc which were positively-related to cancer formation and inhibited the expressions of Rb and alpha 1-I3 which were negatively-related to cancer formation. This implied that tumor promotion played an important role in cancer development and tumor promoters exerted their effects selectively according to the attributes of different genes.

3T3 Cells↗

[Isolation, purification and characterization of human proteinase inhibitor, alpha 2-macroglobulin].

A study of the isolation, purification and characterization of human proteinase inhibitor, alpha 2-macroglobulin (alpha 2-M) is presented. The alpha 2-M, with a purity of more than 90% and subunit molecular weight of 185kDa, was isolated by ammonium sulfate fractionated precipitation from human blood plasma and purified by DEAE cellulose DE52 column chromatography. It was found to be the same as the standard counterpart provided by sigma company. This purification procedure of alpha 2-M can be done with low cost, high efficiency and in large scale preparation.

Chromatography↗