Biomedical subjects

J Bao

Publications and source records attributed to J Bao.

At least 19 recordsLinked to original sources

PubMed2000

Threonine phosphorylation diverts internalized epidermal growth factor receptors from a degradative pathway to the recycling endosome.

Transregulation of the epidermal growth factor receptor (EGFR) by protein kinase C (PKC) serves as a model for heterologous desensitization of receptor tyrosine kinases, but the underlying mechanism remained unknown. By using c-Cbl-induced ubiquitination of EGFR as a marker for transfer from early to late endosomes, we provide evidence that PKC can inhibit this process. In parallel, receptor down-regulation and degradation are significantly reduced. The inhibitory effects of PKC are mediated by a single threonine residue (threonine 654) of EGFR, which serves as a major PKC phosphorylation site. Biochemical and morphological analyses indicate that threonine-phosphorylated EGFR molecules undergo normal internalization, but instead of sorting to lysosomal degradation, they recycle back to the cell surface. In conclusion, by sorting EGFR to the recycling endosome, heterologous desensitization restrains ligand-induced down-regulation of EGFR.

Animals↗

PubMed2000

Enhanced transdermal delivery of tetracaine by electroporation.

The effect of electroporation on the transport of tetracaine through skin in vitro was studied using side-by-side compartment diffusion cells method. After achieving steady state by passive diffusion, fluxes of tetracaine achieved with passive diffusion, electroporative pulse and iontophoresis were compared. Electroporation (square-wave pulse, voltage 130 V, pulse time 0.4 s, pulse frequency 40 pulses min(-1)) or iontophoresis (0.2.mA cm(-2), lasting for 4 h) increased the transport of tetracaine through skin. The flux of tetracaine at 0.25 h after electroporation (pulse number 400) was 54.6+/-6.0 microg.cm(-2).h(-1), that after iontophoresis was 17.4+/-5.8 microg.cm(-2).h(-1) and that after passive diffusion was 8.2+/-0.5 microg.cm(-2).h(-1). In addition, the fluxes of tetracaine increased with the increasing of pulse number. From these results, it is clear that electroporation is effective in enhancing transdermal delivery of tetracaine and its function is better than iontophoresis.

Administration, Cutaneous↗

PubMed2000

Molecular activation of noradrenergic neurons in the rabbit brainstem after coitus.

Our previous studies indicate that coitus in female rabbits induces a gonadotropin-releasing hormone (GnRH) surge that is preceded by an increase in hypothalamic norepinephrine (NE) release. The additional findings of an enhanced tyrosine hydroxylase (TH) mRNA expression in the female brainstem after coitus, in addition to the appropriate topographic distribution of TH and dopamine-beta-hydroxylase (DBH), lead us to hypothesize that coital signals are relayed to hypothalamic GnRH-secreting neurons via brainstem NE-containing perikarya. Here we analyzed coitally activated areas in the brainstem by in situ hybridization of the oncogene c-fos, as well as the expression of TH mRNA at 0, 30 and 60 min postcoitus using specific 35S-labeled probes for c-fos and TH. To establish the identity of activated brainstem neurons, we immunocytochemically double-labeled cells with specific antibodies against Fos protein and DBH at 90 min postcoitus. Both c-fos and TH mRNAs were present at 0 min (control) in the A1, A2 and A6 brainstem-noradrenergic areas. At 30 min after coitus the expression of both genes significantly increased (P<0.01) in the A1 and A2 areas. By 60 min postcoitus the expression of c-fos mRNA decreased to control levels, while that of TH mRNA remained stimulated. Double-labeling of Fos and DBH indicated that the number of dual-labeled neurons increased (P<0.05) over control levels only in the A1 and A2 areas (not in A6) at 90 min postcoitus. These findings support the hypothesis that coitus activates transcriptional/translational events within brainstem NE neurons that culminate in the release of hypothalamic NE and hence a GnRH surge.

Animals↗

PubMed2000

Chronic methylphenidate treatment enhances water maze performance following traumatic brain injury in rats.

Methylphenidate (MPH), a central nervous system stimulant with dopaminergic activity, facilitates neurobehavioral outcome following cortical suction ablation injury, but its potential efficacy following experimental traumatic brain injury (TBI) is unknown. Thus, beginning 24 h after controlled cortical impact injury or sham surgery, male Sprague-Dawley rats were injected (i.p.) once daily for 18 days with either MPH (5 mg/kg) or saline vehicle (VEH) and motor function assessed on post-operative days 1-4, followed by Morris water maze training to find a hidden platform on days 14-18. The MPH treatment regimen was ineffective in accelerating beam-balance or beam-walk recovery, but did significantly decrease swim latencies when compared to VEH-treated controls. The results are consistent with published studies showing improved outcome with MPH therapy. Furthermore, this positive finding with delayed treatment suggests that strategies that enhance catecholamine neurotransmission during the chronic post injury phase may be a useful adjunct in ameliorating some of the neurobehavioral sequelae following TBI in humans.

Analysis of Variance↗

PubMed2000

Stable transfectants of smooth muscle cell line lacking the expression of myosin light chain kinase and their characterization with respect to the actomyosin system.

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666-6671).

Actomyosin↗

PubMed2000

Synergistic effects of olanzapine and other antipsychotic agents in combination with fluoxetine on norepinephrine and dopamine release in rat prefrontal cortex.

To understand the mechanism of the clinical efficacy of olanzapine and fluoxetine combination therapy for treatment-resistant depression (TRD), we studied the effects of olanzapine and other antipsychotics in combination with the selective serotonin uptake inhibitors fluoxetine or sertraline on neurotransmitter release in rat prefrontal cortex (PFC) using microdialysis. The combination of olanzapine and fluoxetine produced robust, sustained increases of extracellular levels of dopamine ([DA](ex)) and norepinephrine ([NE](ex)) up to 361 +/- 28% and 272 +/- 16% of the baseline, respectively, which were significantly greater than either drug alone. This combination produced a slightly smaller increase of serotonin ([5-HT](ex)) than fluoxetine alone. The combination of clozapine or risperidone with fluoxetine produced less robust and persistent increases of [DA](ex) and [NE](ex). The combination of haloperidol or MDL 100907 with fluoxetine did not increase the monoamines more than fluoxetine alone. Olanzapine plus sertraline combination increased only [DA](ex). Therefore, the large, sustained increase of [DA](ex), [NE](ex), and [5-HT](ex) in PFC after olanzapine-fluoxetine treatment was unique and may contribute to the profound antidepressive effect of the olanzapine and fluoxetine therapy in TRD.

Animals↗

PubMed2000

Oestrogen upregulates noradrenaline release in the mediobasal hypothalamus and tyrosine hydroxylase gene expression in the brainstem of ovariectomized rhesus macaques.

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized-oestrogen-supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol-17beta (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge-like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P < 0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2-infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER-alpha and ER-beta mRNAs were expressed in the locus coeruleus, although the expression was greater for ER-alpha than for ER-beta. We conclude that i.v. infusion of E2, which induces a preovulatory surge-like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER-mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2-stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.

Animals↗

PubMed2000

Regulation of neurogenesis by interactions between HEN1 and neuronal LMO proteins.

Basic-helix-loop-helix transcription factors regulate neurogenesis and neuronal differentiation by as yet unknown mechanisms. We show that an embryonic neuronal-specific basic-helix-loop-helix protein, HEN1 (also known as NSCL1 or NHLH), interacts with 'LIM only' proteins. Examination of the expression patterns of XHEN1 and XLMO-3, the Xenopus homologues of these human genes, reveals extensive overlap during early neurogenesis: at the onset of gastrulation on the dorsal side of the blastopore lip and, subsequently, in the prospective neural plate. Binding of XLMO-3 increases the transcriptional activity of XHEN1 in vivo. Co-expression of these two genes in Xenopus embryos induces a cascade of expression of neuronal-specific basic-helix-loop-helix proteins that leads to neuronal differentiation. We propose that XHEN1, in concert with XLMO-3, is a critical regulator of neurogenesis.

Amino Acid Sequence↗

PubMed1999

Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels.

The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.3 channels by margatoxin was previously shown to prevent T cell activation and attenuate immune responses in vivo. In the present study, a triterpene natural product, correolide, was found to block Kv1.3 channels in human and miniswine T cells by electrophysiological characterization. T cell activation events, such as anti-CD3-induced calcium elevation, IL-2 production, and proliferation were inhibited by correolide in a dose-dependent manner. More potent analogs were evaluated for pharmacokinetic profiles and subsequently tested in a delayed-type hypersensitivity (DTH) response to tuberculin in the miniswine. Two compounds were dosed orally, iv, or im, and both compounds suppressed DTH responses, demonstrating that small molecule blockers of Kv1.3 channels can act as immunosuppressive agents in vivo. These studies establish correolide and its derivatives as novel immunosuppressants.

Animals↗

PubMed1998

Differences in Ca2+ channels governing generation of miniature and evoked excitatory synaptic currents in spinal laminae I and II.

Many neurons of spinal laminae I and II, a region concerned with pain and other somatosensory mechanisms, display frequent miniature "spontaneous" EPSCs (mEPSCs). In a number of instances, mEPSCs occur often enough to influence neuronal excitability. To compare generation of mEPSCs to EPSCs evoked by dorsal root stimulation (DR-EPSCs), various agents affecting neuronal activity and Ca2+ channels were applied to in vitro slice preparations of rodent spinal cord during tight-seal, whole-cell, voltage-clamp recordings from laminae I and II neurons. The AMPA/kainate glutamate receptor antagonist CNQX (10-20 microM) regularly abolished DR-EPSCs. In many neurons CNQX also eliminated mEPSCs; however, in a number of cases a proportion of the mEPSCs were resistant to CNQX suggesting that in these instances different mediators or receptors were also involved. Cd2+ (10-50 microM) blocked evoked EPSCs without suppressing mEPSC occurrence. In contrast, Ni2+ (</=100 microM), a low-threshold Ca2+ channel antagonist, markedly decreased mEPSC frequency while leaving evoked monosynaptic EPSCs little changed. Selective organic antagonists of high-threshold (HVA) Ca2+ channels, nimodipine, omega-Conotoxin GVIA, and Agatoxin IVA partially suppressed DR-EPSCs, however, they had little or no effect on mEPSC frequency. La3+ and mibefradil, agents interfering with low-threshold Ca2+ channels, regularly decreased mEPSC frequency with little effect on fast-evoked EPSCs. Increased [K+]o (5-10 mM) in the superfusion, producing modest depolarizations, consistently increased mEPSC frequency; an increase suppressed by mibefradil but not by HVA Ca2+ channel antagonists. Together these observations indicate that different Ca2+ channels are important for evoked EPSCs and mEPSCs in spinal laminae I and II and implicate a low-threshold type of Ca2+ channel in generation of mEPSCs.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1998

Pathogenic poxviruses reveal viral strategies to exploit the ErbB signaling network.

Virulence of poxviruses, the causative agents of smallpox, depends on virus-encoded growth factors related to the mammalian epidermal growth factor (EGF). Here we report that the growth factors of Shope fibroma virus, Myxoma virus and vaccinia virus (SFGF, MGF and VGF) display unique patterns of specificity to ErbB receptor tyrosine kinases; whereas SFGF is a broad-specificity ligand, VGF binds primarily to ErbB-1 homodimers, and the exclusive receptor for MGF is a heterodimer comprised of ErbB-2 and ErbB-3. In spite of 10- to 1000-fold lower binding affinity to their respective receptors, the viral ligands are mitogenically equivalent or even more potent than their mammalian counterparts. This remarkable enhancement of cell growth is due to attenuation of receptor degradation and ubiquitination, which leads to sustained signal transduction. Our results imply that signal potentiation and precise targeting to specific receptor combinations contribute to cell transformation at sites of poxvirus infection, and they underscore the importance of the often ignored low-affinity ligand-receptor interactions.

Animals↗

PubMed1998

Frequent coexpression of MRP/GS-X pump and gamma-glutamylcysteine synthetase mRNA in drug-resistant cells, untreated tumor cells, and normal mouse tissues.

Expression of the multidrug-resistance protein gene MRP, which confers non-P-glycoprotein-mediated multidrug resistance, has been found in many drug-resistant variants and tumor samples. Recent studies have demonstrated that MRP functions as an ATP-dependent transporter functionally related to the previously described glutathione-conjugate (GS-X) pump. We have shown recently that the MRP and gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit mRNA levels are coordinately overexpressed in cisplatin (CP)-resistant human leukemia cells (Ishikawa et al., J Biol Chem 271: 14981-14988, 1996) and frequently co-elevated in human colorectal tumors (Kuo et al., Cancer Res 56: 3642-3644, 1996). In the present study, we showed the coexpression patterns of thirteen additional human drug-resistant cell lines representing different tumor cell origins selected with different agents, except for one doxorubicin-selected line which demonstrated minor elevation in MRP mRNA with no detectable increase in gamma-GCS mRNA, suggesting that the increase of MRP mRNA preceded the increase in gamma-GCS mRNA. Furthermore, in seventeen randomly selected untreated tumor cell lines, the overall correlation coefficient between MRP and gamma-GCS mRNA levels was 0.861. In normal mice, the correlation coefficient of mrp and gamma-gcs mRNA was 0.662 in fourteen tissues (kidney and liver were not included) analyzed. Kidney and liver expressed low levels of mrp relative to gamma-gcs; however, these two tissues expressed high levels of a functionally related mrp homologue, mrp2 (cMoat or cMrp), which may have compensated for the underexpressed mrp in maintaining the total GS-X pump activities. Altogether, these results demonstrated the frequent coexpression of these two genes in various cell settings.

ATP-Binding Cassette Transporters↗

PubMed1998

Comparison of biodegradable and metallic tension-band fixation for patella fractures. 38 patients followed for 2 years.

We compared the outcome of patella fractures fixed by biodegradable tension-band (B) with self-reinforced polyglycolide or self-reinforced poly-L-lactide plugs and polyester ligaments or by metallic tension-band (M) with Kirschner wires and metallic cerclage wire in a randomized study. 38 fractures (18 with B and 20 with M) were treated. The follow-up time was 24 (14-32) months. The fractures healed in all patients after a medium of 8 weeks. In the B group, the clinical outcome was good in 13, fair in 4, and poor in 1 patient. In the M group, the corresponding figures were 15, 3 and 2. There were no clinical or radiographic differences between the two methods. Patella fractures can be treated, successfully using biodegradable tension-band fixation with no need for a second operation to remove the implants after bone union.

Absorption↗

PubMed1998

[Clinical study of gentamycin-loaded chitosan drug delivery system].

An clinical and pharmacokinetic study for a drug delivery system (DDS) of gentamycin-loaded chitosan bar were carried out with the purpose to evaluate its efficacy and giving further data for its clinical applications. Eighteen cases of chronic osteomyelitis were treated by surgical necrectomy with implantation of gentamycin-load chitosan bar in the prepared bone cavity. After operation, the concentration of gentamycin in serum and wound drainage fluid were examined at different times and blood urea nitrogen (BUN) and serum creatinine (Cr) as well. The clinical results were evaluated by the conditions of wound healing and clinical and roentgenographic manifestations. The results showed that the serum gentamycin concentration reached its peak level (0.86 microgram/ml) at 24 hours after operation and lasted for 4 days. No increase in the concentrations of BUN and Cr were observed after implantation. The gentamycin concentration in wound drainage fluid was several hundred times higher than the minimum inhibitory concentration (MIC) for staphylococcus aureus. All of the 18 cases were followed up for 24.8 months (in an range of 6-34 months) 16 patients received initial cure and without any recurrence. So, it could be concluded that the gentamycin-loaded chitosan DDS was a simple and effective method for the treatment of chronic osteomylitis without the necessity to carry out a second operation to remove the drug carrier, and it was sound to popularize its clinical application.

Administration, Topical↗

PubMed1998

TCM differential treatment of child hood persisting pneumonia--a report of 52 cases.

According to TCM differentiation of the symptoms and signs, 52 children with presisting pneumonia were divided into the following four types: 1) deficiency of body fluid due to accumulation of heat in the lung, 2) earth failing to generate metal, 3) deficiency of the Ying and Wei systems, and 4) excessiveness of pathogens due to qi deficiency. After treatment for 2-3 weeks, 40-cases were cured, 8 improved, and 4 ineffective.

Adolescent↗

PubMed1998

Metabolic and dispositional fate of 1,2-dibromo-2,4-dicyanobutane in the male fischer 344 Rat.

Studies were conducted to characterize the absorption, disposition, metabolism, and excretion of 1,2-dibromo-2,4-dicyanobutane (BCB; methyldibromoglutaronitrile) following iv, oral, and topical administration to male Fischer 344 rats. Following iv administration of [14C]BCB (8 mg/kg, 120 microCi/kg), no parent compound was detected in the blood; however, its debrominated metabolite, 2-methyleneglutaronitrile (2-MGN; Cmax 7.3 micrograms/ml), was observed up to 1 hr. Within 72 hr, greater than 60% of the dose was excreted in the urine and 4.1% in the feces, and 6.6% was exhaled as 14CO2. Although less than 5% of the dose was retained in tissues, approximately 12% was bound to the erythrocyte fraction of the blood. Following oral administration of [14C]BCB (80 mg/kg, 100 microgramsCi/kg), approximately 85% of the dose was absorbed, whereas 72% of the dosed radioactivity was recovered in the urine and 9.7% in the feces, 7.5% was exhaled as 14CO2, 3.5% bound to tissues, and 2. 6% bound to blood. Although parent compound could not be detected in the blood following oral administration, 2-MGN was detected (Cmax 0. 32 micrograms/ml). Following topical application of [14C]BCB (25 mg/kg, 50 microgramsCi/kg), less than 12% of the dose was absorbed, with the major route of excretion being the urine (6.6% of dose). Urinary metabolite profiles were nearly identical for each route of administration, and the primary urinary metabolite was a mercapturate conjugate of 2-MGN that was identified as N-acetyl-S-(2, 4-dicyanobutane)-L-cysteine. BCB was found to be extremely labile in whole blood, plasma, and glutathione containing solutions, and in each case the formation of 2-MGN could be reduced by the alkylation of free-sulfhydryls with N-ethylmaleimide. These results suggest that BCB is totally debrominated prior to systemic distribution, and tissue exposure to intact BCB seems to be exceedingly low regardless of route of administration.

Animals↗

PubMed1998

Sulfhydryl-dependent biotransformation and macromolecular binding of 1,2-dibromo-2,4-dicyanobutane in blood.

1,2-Dibromo-2,4-dicyanobutane (BCB) is a broad-spectrum microbicide used commercially in consumer products. The objectives of this study were to elucidate the biotransformation of BCB, characterize its ability to covalently bind macromolecules, and predict the possible toxicological ramifications of such events. After iv administration of [14C]BCB to male Fischer 344 rats, 14C-equivalents were observed to bind gradually to blood constituents. By 48 hr, approximately 12% of the total dose was covalently bound. At no time was parent compound detected in the blood. However, the debrominated BCB metabolite 2-methyleneglutaronitrile (MGN) was observed. In vitro experiments revealed that BCB was extremely labile and was readily debrominated in fresh whole blood, erythrocyte preparations, and buffered glutathione (GSH) solutions. In each case, the formation of MGN was inhibited by the alkylation of free sulfhydryls with N-ethylmaleimide (NEM). For every 1 mol of BCB converted to MGN, 2 mol of GSH were oxidized to glutathione disulfide (GSSG) (BCB + 2 GSH --> MGN + GSSG + 2 HBr). The oxidation of free sulfhydryls during the conversion of BCB to MGN caused erythrocyte hemolysis (EC50 approximately 1 mM) in isolated preparations. Hemolysis was increased by coincubation of BCB with NEM (EC50 approximately 0.3 mM) and was decreased by coincubation with GSH (EC50 > 3 mM). However, MGN did not cause hemolysis of erythrocytes, even at concentrations 10-fold higher than the EC50 of BCB. In vitro experiments also demonstrated that incubation with either BCB or MGN resulted in significant macromolecular binding to the erythrocyte fraction of the blood (approximately 80%). Incubation with NEM resulted in a significant decrease in binding for both BCB (11.3% bound) and MGN (29.5% bound). Because BCB is rapidly debrominated in whole blood, it appears that MGN is the reactive species responsible for macromolecular binding. From these studies, we conclude that the conversion of BCB to MGN is mediated by a free sulfhydryl-dependent biotransformation pathway. Furthermore, BCB biotransformation is required for erythrocyte binding, and the consumption of free sulfhydryls associated with the biotransformation of BCB is responsible for hemolysis.

Animals↗

PubMed1997

Microelectrode arrays for stimulation of neural slice preparations.

A planar 6 x 6 array of iridium electrodes with four reference electrodes has been developed for use with neural tissue preparations. Precise knowledge of the relative locations of the array elements allows for spatial neurophysiological analyses. The 10 microns diameter platinized iridium electrodes on a 100 microns pitch have been used to stimulate acutely prepared slices of spinal cord from free-ranging rodents. An intracellular recording from a single neuron in the substantia gelatinosa (SG) using the whole-cell, tight-seal technique allowed low noise, high resolution studies of excitatory or inhibitory electrical responses of a given neuron to inputs from the primary afferent fibers or from stimulation by individual electrodes of the array. The resulting maps of responses provide an indication of the interconnectivity of neural processes. The pattern emerging is that of limited interconnectivity in the SG from areas surrounding a recorded neuron but with strong excitatory or inhibitory effects from those oriented in a longitudinal (rostral-caudal) direction relative to the neuron. The observations to date suggest the neurons of the SG are arranged in sets of independent networks, possibly related to sensory modality and input from particular body regions.

Animals↗