Biomedical subjects

J Y Chang

Publications and source records attributed to J Y Chang.

At least 55 records · Page 3Linked to original sources

PubMed1998

Unfolding and refolding of cardiotoxin III elucidated by reversible conversion of the native and scrambled species.

Cardiotoxin analogue III (CTX III) isolated from the venom of the Taiwan Cobra (Naja naja atra) is a small molecular weight, all beta-sheet protein, cross-linked by four disulfide bridges. The unfolding and refolding mechanisms of CTX III have been examined by monitoring the reversible conversion of the native and scrambled species. It is found that, in the presence of a denaturant (urea/guanidinium hydrochloride) and a thiol catalyst, CTX III forms a mixture of scrambled species by shuffling its four native disulfide bonds. Complete unfolding of CTX III can be achieved using either 3.0-4.0 M guanidinium hydrochloride (GdmCl) or 5.0-6.0 M urea. It is observed that GdmCl is thermodynamically more potent but kinetically less efficient than urea in unfolding CTX III. The rate constants of unfolding of CTX III in 8 M urea are significantly greater than that obtained in 5.0 M GdmCl and 8.0 M GdmCl. Interestingly, upon removal of the denaturant, scrambled species of CTX III is found to refold spontaneously through dynamic reshuffling of the non-native disulfides to attain the native disulfide linkages. In addition, CTX III contains highly reactive lysines which are modified by trace amounts of cyanate contaminant which exists invariably even in high-grade urea solutions. The reactive lysines of CTX III are modified by cyanate both in the native and unfolded states of the protein. The modification is nonselective, and the modified product is found to consist of highly heterogeneous species. Surprisingly, these heterogeneous species of modified CTX III are observed to display stability and folding/unfolding properties indistinguishable from those of the native CTX III. The knowledge obtained from the present study, on the conditions to convert the scrambled species, could provide useful clues for a rational design for snake venom cardiotoxins with potential therapeutic applications.

Animals↗

PubMed1998

Comparison of mesocorticolimbic neuronal responses during cocaine and heroin self-administration in freely moving rats.

To compare neuronal activity within the mesocorticolimbic circuit during the self-administration of cocaine and heroin, multiple-channel single-unit recordings of spike activity within the rat medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) were obtained during the consecutive self-administration of cocaine and heroin within the same session. The variety of neuronal responses observed before the lever press are termed anticipatory responses, and those observed after the lever press are called post-drug infusion responses. For the total of the 110 mPFC and 111 NAc neurons recorded, 30-50% of neurons, depending on the individual sessions, had no alteration in spike activity in relation to either cocaine or heroin self-administration. Among the neurons exhibiting significant neuronal responses during a self-administration session, only a small portion (16-25%) of neurons responded similarly under both reinforcement conditions; the majority of neurons (75-84%) responded differently to cocaine and heroin self-administration as revealed by variations in both anticipatory and/or post-drug infusion responses. A detailed video analysis of specific movements to obtain the self-administration of both drugs provided evidence against the possibility that locomotive differences contributed to the observed differences in anticipatory responses. The overall mean activity of neurons recorded in mPFC and NAc measured across the duration of the session segment for either cocaine or heroin self-administration also was different for some neurons under the two reinforcement conditions. This study provides direct evidence that, in mPFC and NAc, heterogeneous neuronal circuits mediate cocaine and heroin self-administration and that distinct, but overlapping, subpopulations of neurons in these areas become active during operant responding for different reinforcers.

Action Potentials↗

PubMed1998

Addition of aprotinin to organ preservation solutions decreases lung reperfusion injury.

BACKGROUND: Organ preservation injury is associated with endothelial cell damage, destabilization of mitochondrial and cell membranes, and the release of proteolytic enzymes. In addition to its well-known clinical effect of reducing perioperative blood loss, aprotinin has antiproteolytic and membrane-stabilizing properties. We hypothesized that adding aprotinin to Euro-Collins (EC) and University of Wisconsin (UW) solutions would decrease preservation injury in cultured endothelial cells and a whole organ rat lung model. METHODS: Bovine aortic endothelial cells were cultured and stored in the respective solution at 4 degrees C for 12 or 48 hours. Endothelial cell viability after storage was assessed by dimethylthiazole tetrazolium cytotoxicity assay. In the whole organ model, rat lungs were isolated, flushed with the respective solution, and stored at 4 degrees C for 6 or 12 hours. The lungs were ventilated with 100% O2 and reperfused with fresh blood. Alveolar-arterial O2 difference, O2 tension, capillary filtration coefficient, and compliance were determined. RESULTS: Endothelial cell viability was optimized with the addition of aprotinin to EC and UW at a dose of 150 KIU/mL (0.02 mg/mL). In the isolated perfused lung model, after 6 hours of ischemic storage, aprotinin-enhanced (100 KIU/mL [0.014 mg/mL]) EC and UW decreased alveolar-arterial O2 difference, increased O2 tension, and decreased capillary filtration coefficient compared with EC and UW alone. After 12 hours of ischemic storage, aprotinin-enhanced EC and UW decreased alveolar-arterial O2 difference, increased O2 tension, decreased capillary filtration coefficient, and increased compliance compared with EC and UW alone. CONCLUSIONS: The addition of aprotinin to EC and UW solutions increases endothelial cell viability in hypoxic cold storage conditions. In terms of whole organ function, aprotinin improves lung preservation as demonstrated by increased oxygenation and compliance, and decreased capillary permeability. This study is clinically applicable as there is already extensive experience with the use of aprotinin in heart and lung transplant recipients, in addition to its routine use in conventional cardiac operations.

Adenosine↗

PubMed1998

Aliphatic aldehydes and allethrin in mosquito-coil smoke.

Presence of aliphatic aldehydes and allethrin in the smoke produced by two brands of mosquito-coil was determined by high performance liquid chromatography. It was observed that burning mosquito-coil produces a greater amount of formaldehyde, acetaldehyde and acrolein in the gaseous phase, whereas lesser amount of particulate-bounded aldehydes. Aldehydes bounded in particulates were, however, enriched in terms of concentration. Allethrin was found to occur only in particulates of 0.1-1.0 micron size. The results reveal that allethrin may not be capable of attacking the mosquitoes effectively.

Air Pollutants↗

PubMed1998

Neurotoxicity of cholesterol oxides on cultured cerebellar granule cells.

Cultured rat cerebellar granule cells were used to determine the potential neurotoxicity of cholesterol oxides. The cholesterol oxides tested included: 7-beta-OH-, 7-keto-, 19-OH-, 22(R)-OH-, 22(S)-OH- and 25-OH- cholesterol. Among them, 7-beta-OH- and 7-keto-cholesterol were the most efficacious in causing neuronal death such that 20 microg/ml (50 microM) of these agents killed more than 80% of cells in 2 days. 7-beta-OH-cholesterol at this concentration killed 50% of cells in approximately 7 h. A number of pharmacological agents were tested for their abilities to prevent neuronal death induced by cholesterol oxides. Among them, aurintricarboxylic acid, vitamin E and methyl-beta-cyclodextrin were able to prevent cholesterol oxide-induced neurotoxicity in a dose-dependent manner. These results suggest that, in addition to causing pathological changes in cells directly involved in atherosclerosis, cholesterol oxides may induce toxicity in neurons of the central nervous system.

Animals↗

PubMed1998

Neurotoxicity of 25-OH-cholesterol on sympathetic neurons.

Cultured rat sympathetic neurons derived from postnatal rat superior cervical ganglia (SCG) were used to compare the neurotoxicity of several cholesterol oxides. The cholesterol oxides tested included: 7-beta-OH-, 7-keto-, 19-OH-, 22(R)-OH-, 22(S)-OH-, and 25-OH-cholesterol. These agents caused an acute as well as a delayed toxicity in sympathetic neurons with 25-OH-cholesterol appearing to be the most toxic. A time-dependent experiment indicated that 25-OH-cholesterol at 4 microg/ml (10 microM) was able to kill 50% of the cells in 36 h. Morphological studies indicate that most of the cells do not exhibit a structural change similar to that observed in neuronal programmed cell death. Whole-cell patch clamp recording of untreated controls and 25-OH-cholesterol (2 microg/ml)-treated cells indicated that this toxicity was not accompanied by significant changes in voltage-dependent calcium channel activity. A number of pharmacological agents including ethylene glycolbis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), cycloheximide, KCl, vitamin E, and methyl-beta-cyclodextrin were able to prevent the 25-OH-cholesterol-induced cell death to various degrees. These results suggest that, in addition to causing pathological changes in cells directly involved in atherosclerosis, cholesterol oxides may induce neurotoxicity in sympathetic neurons.

Animals↗

PubMed1998

Neurotoxicity of 25-OH-cholesterol on NGF-differentiated PC12 cells.

PC12 cells induced to differentiate with nerve growth factor were used to study the neurotoxicity of 25-OH-cholesterol. This agent induced a dose- and time-dependent cell death in neuronal PC12 cells. Cells treated with this agent showed condensed nuclei, a morphology similar to that of cells dying of programmed cell death. However, agents known to prevent neuronal programmed cell death (cyclic AMP, KCl, aurintricarboxylic acid, and cycloheximide) failed to prevent the 25-OH-cholesterol-mediated cytotoxicity. On the other hand, cell death induced by 25-OH-cholesterol was prevented by treatment with vitamin E and methyl-beta-cyclodextrin. In contrast to observations made in other cell types, whole-cell patch clamp recording of neuronal PC12 cells revealed that treatment with 25-OH-cholesterol did not significantly alter calcium influx through voltage-dependent channels. These results provide the first characterization of the toxicity of cholesterol oxides toward neuronal PC12 cells, which should be useful in future studies on the interactions between cholesterol oxides and cells from the nervous system.

Animals↗

PubMed1998

Safety study and characterization of E1A-liposome complex gene-delivery protocol in an ovarian cancer model.

A phase I clinical trial of E1A-liposome complex is currently ongoing in patients with HER-2/neu-overexpressing breast or ovarian cancers. To optimize the E1A-liposome complex for a further stage of clinical trial, several aspects of the current protocol have been examined in an animal model. In the orthotopic ovarian cancer model, different doses of lipid in the the E1A-liposome complex, which is currently used in clinical trials, were tested for the in vivo gene-transfer efficacy and tumor-suppression function. A lowered lipid dose--1/13 of the previous amount--produced gene expression level and E1A tumor-suppression efficacy similar to that of the original protocol. Mini-E1A, an E1A construct without its immortalization domain and yet capable of repressing HER-2/neu, was proved to be as potent as E1A in suppressing tumor development in vivo. These changes in the E1A-liposome complex will significantly reduce any potential adverse effects caused by lipid vector and E1A DNA. To examine further whether residual E1A DNA may still exist in normal organs after the E1A-liposome treatment, PCR was used to detect E1A DNA in mice that survived for 1 1/2 years after the last treatment. E1A DNA was detected only in the lungs and kidneys, but not in livers, hearts, spleens, brains, uterus or the ovaries. Furthermore, resistance of the E1A DNA extracted from tissues to the digestion of Dpnl restriction enzyme, which can cleave the methylated E1A plasmid DNA generated by methylation-competent bacteria, suggested integration of E1A DNA into the chromosome of the lungs and kidneys. Experimental results presented here provide important information for safety concerns and for the design of future phase II and phase III trials.

Adenovirus E1A Proteins↗

PubMed1998

Newer concepts of blood coagulation.

In this report we describe an in vitro model of blood coagulation reactions that mimics as closely as possible the in vivo condition. Our model indicates that the tissue factor-factor VIIa complex initiates coagulation by activating small amounts of both factor IX and factor X in the environment of the tissue factor bearing cell. Factor Xa and factor IXa formed in the initial reaction then play very distinct roles in the subsequent interactions of the clotting mechanism leading to a burst of thrombin generation on the platelet surface. Our results also indicate that factor XI can be activated by thrombin in the absence of factor XII and that the function of factor XI is simply to enhance conversion of factor IX to factor IXa resulting in enhanced thrombin generation on the platelet surface.

Animals↗

PubMed1998

Toxicity of cholesterol oxides on cultured neuroretinal cells.

PURPOSE: By using nerve growth factor-differentiated PC12 cells as a model for sympathetic neurons, we have recently shown that cholesterol oxides are toxic to cells of neural origin. Since lipid metabolism is known to be involved in some pathological conditions associated with the visual system, we sought to extend this line of research by studying the potential cytotoxicity of cholesterol oxides on primary cultures derived from neuroretinas. METHODS: Dissociated cultures derived from neuroretinas of 1-day-old Sprague-Dawley rats were used in this series of studies. Immunohistochemical staining was used to identify neuronal and glial cell types in these cultures. MTT assay was used to determine the cytotoxicity of cholesterol oxides, including 7-beta-OH-, 7-keto-, 19-OH-, 22(R)-OH-, 22(S)-OH- and 25-OH-cholesterol. RESULTS: Among the cholesterol oxides tested, 7-beta-OH- and 7-keto-cholesterol were the most effective in causing cell death, such that 20 micrograms/ml (50 microM) of these agents killed approximately 80% of cells in 3 days. A time-dependent experiment indicated that 10 micrograms/ml of 7-beta-cholesterol was able to kill 50% of cells in approximately 5 h. A number of pharmacological agents were tested for their ability to prevent cell death induced by cholesterol oxides. Among them, vitamin E and methyl-beta-cyclodextrin were able to prevent cholesterol oxide-induced cell death in a dose-dependent manner. CONCLUSIONS: These results suggest that, in addition to causing pathological changes in cells directly involved in atherosclerosis, cholesterol oxides may be toxic to cells derived from neuroretinas.

Animals↗

PubMed1998

Cloning, expression, and characterization of mouse tissue factor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

Amino Acid Sequence↗

PubMed1998

Ethanol action on neural networks studied with multineuron recording in freely moving animals.

The advent of new chronic multineuron recording techniques for examining neural activity in behaving animals has initiated a new phase in the analysis of the neuronal mechanisms that underlie ethanol and other drug self-administration. The technique allows for the simultaneous recording of groups of individual neurons in one or more brain regions during ongoing behavior; therefore, the spatio-temporal patterns of neuronal activity during specific behavioral events can be determined. We have successfully applied this technique to rat models of cocaine and heroin self-administration. Recently, using rats, we have been able to record from neurons in areas of the mesocorticolimbic circuit during ethanol-reinforced operant responding. In this review, we describe the current and future application of this new behavioral neurophysiology to the investigation of the neurobiology of alcohol addiction.

Alcoholism↗

PubMed1998

Acquired hemophilia A: report of two cases.

Two patients who presented with active bleeding and were diagnosed with acquired hemophilia A (AHA) are reported herein. One was a 27-year-old woman who experienced spontaneous oozing from an episiotomy wound six days after her second normal delivery. Bleeding became progressively worse, despite treatment with primary sutures and curettage of the uterus at a local hospital. She underwent emergency exploratory laparotomy because of intra-abdominal bleeding, during which perforations of the uterus were discovered. Unremitting bleeding from the surgical wound occurred after surgery. The patient was finally diagnosed with AHA when Factor VIII (FVIII) inhibitor (titer, 19 Bethesda units (BU)/ml) was detected in her plasma. She died of refractory hemorrhaging, despite intensive treatment with Factor IX concentrate infusion and cyclophosphamide therapy. The second patient was a 22-year-old man who sustained spontaneous and recurrent intramuscular hemorrhage in the right thigh for one month. Laboratory studies including complete blood count, biochemical evaluation, coagulation screening and immunologic assays were all within normal limits, except for a prolonged activated partial thromboplastin time. Idiopathic AHA was diagnosed after the detection of plasma FVIII inhibitor with a concentration of 5.9 BU/ml. The patient's coagulopathy was successfully managed with plasma exchange and subsequent treatment with oral prednisolone and cyclophosphamide.

Adult↗

PubMed1997

Establishment of two morphologically distinct PC12 cell lines resistant to 25-OH-cholesterol toxicity.

Two novel populations of spontaneous PC12 cell mutants resistant to a toxic concentration of 25-OH-cholesterol (5 microg/ml, 12.5 microM) were isolated and designated as R25R and F25R based on cell morphology. R25R consisted of round cells that were morphologically similar to the parent PC12 cells, and responded to nerve growth factor by extending neurites. F25R was a group of process-bearing flat cells that did not assume a neuronal morphology in the presence of nerve growth factor. These two cell lines also acquired some cross-resistance toward other cholesterol oxides. Nerve growth factor induced prominent voltage-dependent calcium currents in parent PC12 cells and in R25R, but not in F25R. Further experiments indicated that the parent PC12 cells, R25R and F25R exhibited different properties when challenged with a variety of toxic insults, including amphotericin B, serum withdrawal and beta-amyloid protein treatment.

Amyloid beta-Peptides↗

PubMed1997

Transforming growth factor-beta2 selectively alters the developmental expression of the fast transient A-current in cultured rat superior cervical ganglion neurons.

Cultures of neonatal rat superior cervical ganglion (SCG) were utilized to examine the ability of transforming growth factor-beta2 (TGFbeta2) to alter voltage-gated K+ channel development. Whole-cell patch clamp recordings were used to monitor changes in three separate K+ currents: A rapidly inactivating A-current (I(Af)), a slowly inactivating A-current (I(As)), and a non-inactivating current (I(K)). Continuous TGFbeta2 (10 ng/ml) treatment selectively altered the normal developmental decrease in I(Af) expression in SCG neurons, but did not significantly change I(As) or I(K) expression. After 2 weeks of treatment, the mean I(Af) current density in control cultures had decreased 67%, while the I(Af) current density in TGFbeta2 treated cultures remained near initial values (approximately 2.7-fold higher than control). This difference remained even after 4 weeks of exposure. TGFbeta2 did not appear to change the activation kinetics or voltage-dependence of I(Af). These findings indicate that TGFbeta2 may play an important role in modulating the development of neuronal excitability by regulating the expression of voltage-gated K+ channels.

Animals↗

PubMed1997

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.

Amino Acid Chloromethyl Ketones↗

PubMed1997

Neuronal responses in prefrontal cortex and nucleus accumbens during heroin self-administration in freely moving rats.

Chronic multi-channel single unit recordings of neuronal responses in prefrontal cortex (PFC) and nucleus accumbens (NAc) were made in 9 male Sprague Dawley rats to determine patterns of neuronal activity during heroin self-administration. Up to 32 neurons were recorded simultaneously in these two brain regions while rats lever pressed on a continuous reinforcement schedule for intravenous infusion of heroin (30 microg/kg/infusion). The variety of neuronal responses observed before and after each self-administered heroin infusion can be classified according to the following categories: (1) neurons that increased or (2) decreased their activity immediately before the lever press; (3) neurons that increased or (4) decreased their activity after the heroin infusion; and, (5) neurons that did not alter their activity either before or after the lever press for heroin infusion. The majority (69% in the PFC and 65% in the NAc) of neurons sampled fell into this last category of no change, indicating that a selected fraction becomes active during this specific task. In general, NAc neurons displayed more post-heroin responses than PFC neurons while the proportion of neurons showing responses before the lever press was similar in the mPFC and the NAc. This initial description of the responses of PFC and NAc neurons during heroin self-administration suggests that the neuronal circuit of the mesocorticolimbic system is involved in heroin self-administration. This circuit appears to contribute both to the initiation of drug-seeking behavior (pre-lever press phasic neuronal responses), as well as the action of heroin infusion itself (post-infusion phasic neuronal responses) by activation of different subsets of neurons.

Analysis of Variance↗

PubMed1997

The tumor suppression activity of E1A in HER-2/neu-overexpressing breast cancer.

The HER-2/neu proto-oncogene is frequently amplified or overexpressed in human breast and ovarian cancers, and is significantly correlated with shorter survival. We have previously reported that the adenovirus type 5 early region 1A (E1A) gene product can repress HER-2/neu overexpression by repressing HER-2/neu promoter activity, and suppress the tumorigenic potential of HER-2/neu-overexpressing ovarian cancer cells. To examine E1A tumor suppressor function in breast cancer, we transduced E1A in vitro by adenovirus into both HER-2/neu-overexpressing and low expressing human breast cancer cell lines. In HER-2/neu-overexpressing cells, E1A greatly inhibited tumor cell growth in vitro. However, in HER-2/neu low expressing cancer cell lines, E1A had no significant effect on cell growth in culture medium. To test the therapeutic efficacy of E1A, we used both adenovirus-mediated and cationic liposome-mediated E1A gene delivery systems in an orthotopic breast cancer animal model. An advanced breast cancer model was established by inoculation of HER-2/neu-overexpressing human breast cancer cells in mammary fat pad and treated by local injections of either replication-deficient adenovirus expressing E1A, Ad.E1A(+) or a liposome-E1A DNA complex. As controls, mice bearing tumors were also treated with Ad.E1A(-) which is virtually the same adenovirus as Ad.E1A(+) except that E1A is deleted, a liposome-E1A frame-shift mutant DNA complex, or just PBS. In mice bearing a HER-2/neu-overexpressing breast cancer cell line, E1A delivered either by adenovirus or liposome significantly inhibited tumor growth and prolonged mouse survival compared with the controls. In fact, 60-80% of E1A-treated mice lived longer than 2 years versus only 0-20% of control mice (P<0.05). Western blot analysis showed that E1A protein was expressed in tumor tissue and immunohistochemical analysis showed that HER-2/neu p185 protein expression was suppressed. Taken together, our results indicated that both adenovirus and cationic liposome delivery systems were effective in transfering E1A gene for tumor suppression in a HER-2/neu-overexpressing breast cancer model.

Adenovirus E1A Proteins↗