Biomedical subjects

X J Yu

Publications and source records attributed to X J Yu.

At least 73 records · Page 4Linked to original sources

PubMed1995

Barium block of the muscarinic potassium current in guinea-pig atrial cells.

Block of the muscarinic K+ current (iK,ACh) by Ba2+ has been studied in guinea-pig atrial cells using the whole-cell patch-clamp technique. The dose-response curve for the block of iK,ACh can be fitted assuming that a muscarinic K+ channel is blocked when a single Ba2+ ion binds to it (apparent dissociation constant, Kd = 125 microM at 0 mV). Block was voltage and time dependent. The voltage dependence can be explained by Ba2+ binding to a site within the pore of the channel, 36% across the width of the membrane electric field (from the outside). Raising the bathing K+ concentration reduced Ba2+ block of iK,ACh, which suggests that Ba2+ and K+ compete for a common binding site. When Ba2+ was added during an exposure to ACh (muscarinic K+ channel open), block of iK,ACh developed rapidly, but when Ba2+ was added prior to an exposure to ACh (muscarinic K+ channel closed), little block of iK,ACh was evident when ACh was first applied. This suggests that when the muscarinic K+ channel is closed in the absence of ACh, Ba2+ does not have access to the binding site within the pore of the channel. In conclusion, Ba2+ block of iK,ACh is concentration, voltage, time, K+ and state dependent.

Animals↗

PubMed1995

Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells.

Infection by murine retroviruses in embryonic carcinoma (EC) and embryonic stem cells is highly restricted. The transcriptional unit of the Moloney murine leukemic virus (MoMuLV) long terminal repeat (LTR) is inactive in EC and embryonic stem cells in association with increased proviral methylation. In this study, expression in F9 EC cells was achieved from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the myeloproliferative sarcoma virus LTR, substitution of the primer binding site, and either deletion of a negative control region at the 5' end of the LTR or insertion of a demethylating sequence. We conclude that inhibition of expression from the MoMuLV LTR in EC cells is mediated through the additive effects of multiple cis-acting elements affecting the state of methylation of the provirus.

3T3 Cells↗

PubMed1994

Increase of an endogenous inhibitor of nitric oxide synthesis in serum of high cholesterol fed rabbits.

In the present study, the concentration of NG,NG-dimethylarginine (DMA), an endogenous inhibitor of nitric oxide synthesis, was measured with high-performance liquid chromatography, and the effect of hypercholesterolemia on DMA level was investigated in the high fat, high cholesterol fed rabbit. After 6 weeks on a high fat, high cholesterol diet, serum total cholesterol, triglycerides, and lipid peroxides were increased, and atherosclerosis was shown by means of morphological examination. In these rabbits with atherosclerosis, serum level of DMA was significantly raised, while serum creatine level remained normal. This study suggests that chronic hypercholesteremia may stimulate DMA production through elevation of lipid peroxides. The present results implicate endogenous DMA as a contributor to the development of atherosclerosis.

Animals↗

PubMed1994

Protective effect of tetramethylpyrazine against damages of aortic endothelial cells elicited by low-density lipoproteins.

Effects of tetramethylpyrazine (TMP) on endothelial cells damaged by low-density lipoproteins (LDL) were investigated. When endothelial cells were incubated with LDL (1.5 mg protein.ml-1) the level of malondialdehyde (MDA) was increased and the activity of superoxide dismutase (SOD) was inhibited, and levels of cGMP and epoprostenol were decreased. TMP at concentrations of both 20 and 150 mg.L-1 nullified the inhibition of SOD activity and the reduction of cGMP and epoprostenol content elicited by LDL. However, the elevation of MDA content induced by LDL was negated by TMP only at 150 mg.L-1. TMP also caused a reduction in MDA content and an increase of epoprostenol level in normal endothelial cells. This study suggests that TMP protects endothelial cells against damages elicited by LDL and that the protection of TMP might be due to reduction in lipid peroxidation through stimulation of production and/or release of epoprostenol.

Animals↗

PubMed1994

Electrophysiologic effects of total flavones of Hippophae rhamnoides L on guinea pig papillary muscles and cultured rat myocardial cells.

The effects of total flavones of Hippophae rhamnoides L (TFH) were evaluated using conventional microelectrode technic. After administration of TFH 100-200 mg.L-1, the action potential duration of 50% repolarization (APD50) was shortened both in cultured rat myocardial cells and in guinea pig papillary muscles. The slope of phase 4 of depolarization (SP4) in cultured rat myocardial cells was decreased and the contractile force (CF) in guinea pig papillary muscles was weakened. Arrhythmias evoked by strophantin G in guinea pig papillary muscles were suppressed by TFH 100 mg.L-1. These findings suggested that the influence of TFH on myocardial cells may be resulted mainly from its inhibition of Ca2+ influx and its interference with intracellular Ca2+ reservoir.

Action Potentials↗

PubMed1993

The regulatory effect of bradykinin on the actions of sensory nerves in the perfused rat mesentery is mediated by nitric oxide.

In the perfused rat mesentery, when adrenergic nerves were blocked with guanethidine and vascular smooth muscle tone was increased with methoxamine, transmural field stimulation caused a dilator response. Bradykinin significantly suppressed vasodilator responses to a transmural field stimulation in a concentration-dependent manner. After pretreatment with saponin to damage endothelial cells, bradykinin still suppressed vasodilator responses to transmural field stimulation. The effect of bradykinin was unaltered by indomethacin. N omega-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, abolished the inhibition of vasodilator responses to transmural field stimulation elicited by bradykinin. However, in the presence of L-arginine and L-NAME the inhibitory effect of bradykinin reappeared. Furthermore, methylene blue itself caused potentiation of vasodilator responses to transmural field stimulation and reversed the effect of bradykinin. These findings suggest that bradykinin can produce an inhibitory modulation of the actions of sensory nerves in the perfused rat mesentery and that the effect of bradykinin may be mediated by nitric oxide released from non-adrenergic, non-cholinergic nerves.

Animals↗

PubMed1993

Nitric oxide modulates responses to sensory nerve activation of the perfused rat mesentery.

The modulatory actions of nitric oxide on sensory nerves were investigated on dilator responses of the perfused rat mesentery to transmural nerve stimulation. N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, caused a significant augmentation of vasodilator responses to transmural nerve stimulation, an effect which was abolished by L-arginine. L-NAME had no effect on vasodilator responses to exogenous calcitonin gene-related peptide. In preparations without endothelium L-NAME still caused potentiation of vasodilator responses to transmural nerve stimulation. Methylene blue, an inhibitor of guanylate cyclase, also significantly enhanced vasodilator responses to transmural nerve stimulation. After pretreatment with diethyldithiocarbamate to inhibit superoxide dismutase, vasodilator responses to transmural nerve stimulation were also potentiated. This response was abolished by exogenous superoxide dismutase. These findings suggest that endogenous nitric oxide modulates, in an inhibitory fashion, the actions of sensory nerves in the rat mesentery. The results also suggest that endogenous superoxide dismutase may participate in the regulation of the actions of sensory nerves via control of cellular superoxide anion level.

Animals↗

PubMed1993

Aging enhances synaptic efficacy in a latent motor pathway following spinal cord hemisection in adult rats.

The effect of aging on the development of the neural circuitry mediating the crossed phrenic phenomenon (CPP) was investigated in anesthetized, vagotomized, artificially ventilated young adult (9-10 weeks old) and older adult (9-10 months old) rats. Cervical spinal cord hemisection rostral to the phrenic nucleus abolishes respiratory activity in the ipsilateral phrenic nerve. The respiratory activity can be restored by subjecting the animal to respiratory stress after spinal cord injury. The stress activates a normally latent respiratory motor pathway which mediates the functional recovery in the phrenic nerve ipsilateral to hemisection. This is the CPP and the recovered activity in the phrenic nerve is designated, "crossed phrenic activity." In the present study, blood pressure and end-tidal CO2 concentration were monitored. Crossed phrenic activity was induced by asphyxia (turning off the ventilator within 30 min following a left C2 spinal cord hemisection) and recorded from the left phrenic nerve. The mean area under the three largest successive integrated waveforms of the phrenic nerve bursts served as our quantitative assessment of the CPP. The results showed that the mean integrated area in the young rat group was 10.79 +/- 1.58 mm2. The mean integrated area in the older adult rats was significant greater (P < 0.05) at 41.58 +/- 10.05 mm2. Thus, there is almost a fourfold enhancement of crossed phrenic nerve activity that can be generated in older adult rats as compared to young adult rats. In addition, the CPP persists for significantly longer periods during progressive asphyxial hypoxia in the older adult rats than in the young adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

On the role of G protein activation and phosphorylation in desensitization to acetylcholine in guinea-pig atrial cells.

1. The ACh-activated K+ current (IK,ACh) has been investigated in guinea-pig atrial cells at 36 degrees C using the whole-cell patch-clamp technique. 2. During an exposure to ACh, IK,ACh faded as a result of desensitization. Throughout the fade of the current, the current reversed at EK and showed inward-going rectification. The fade was, therefore, the result of a genuine decrease in IK,ACh. 3. The onset of desensitization (as judged by the fade of IK,ACh) was biphasic and the time constants of the fast and slow phases of desensitization were 1.58 +/- 0.14 (n = 16) and 148.2 +/- 12.8 s (n = 18) respectively. Recovery from the fast and slow phases of desensitization (after 30 s and 5 min exposures to ACh respectively) occurred with time constants of 52 and 222 s respectively. This suggests that two processes are involved in desensitization. 4. The Q10 of the rate constant of the fast phase of desensitization was 2.2 +/- 0.3 (n = 6). 5. Intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or extracellular perfusion with AlF4- were used to bypass the muscarinic receptor and trigger IK,ACh by directly activating the G protein, GK, that links the muscarinic receptor to the K+ channel. Both GTP gamma S and AlF4- activated a current with the same reversal potential and the same degree of inward-going rectification as the ACh-activated current. 6. Desensitization still occurred when the muscarinic receptor was bypassed and IK,ACh was triggered by direct activation of GK with either GTP gamma S or AlF4-. This suggests that desensitization is, in part, the result of a modification of either GK or the K+ channel. 7. Activation of the muscarinic receptor by ACh resulted in greater desensitization than direct activation of GK; at the end of a 5 min exposure to ACh, current was only 22 +/- 1% (n = 19) of its peak value, whereas, after direct activation of GK by GTP gamma S for 5 min, current was 42 +/- 6% (n = 5) of its peak value. This suggests that desensitization also involves the muscarinic receptor. 8. When cells were perfused with GTP gamma S, the fast phase of desensitization could still occur, but the slow phase was reduced. This suggests that the fast phase involves GK or the K+ channel, whereas the slow phase involves the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗

PubMed1993

Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes.

trans-Dominant inhibitory mutant versions of the human immunodeficiency virus type 1 (HIV-1) regulatory genes tat and rev have previously been described. We have constructed a series of retroviral vectors to transduce these genes and compare their inhibitory activities. The inhibitory activities were measured with transient transfection assays by using a reporter which expresses an HIV-1 gag-Escherichia coli lacZ fusion protein with strict dependence on coexpression of both tat and rev. Additionally, the vectors were packaged as amphotropic virions and used to stably transduce human CEM T lymphocytes. The transduced CEM cells were challenged with HIV-1, and the effects of the mutant HIV-1 genes were determined by measuring the levels of HIV-1 p24gag produced. A tat gene substituted at amino acid 41 (tatk41a) retained partial trans-activating activity and lacked inhibitory activity. A tat gene with a premature stop codon at amino acid 54 (tat54ter) showed moderate trans-dominant inhibition of the reporter plasmid but failed to significantly inhibit HIV-1 replication. The M10 rev mutant, with a 2-amino-acid substitution, showed strong trans-dominant inhibitory activity both in the reporter plasmid and in the HIV-1 infection assay. The greatest inhibition of HIV-1 growth was seen when M10 was expressed under the transcriptional control of a human cytomegalovirus promoter; slightly less inhibition was achieved when expression of M10 was controlled by the Moloney murine leukemia virus long terminal repeat, and minimal inhibition was seen when the HIV-1 long terminal repeat controlled the M10 gene. These results demonstrate the potential utility of retroviral vectors expressing trans-dominant inhibitory mutant HIV-1 genes for gene therapy approaches to AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1993

A direct negative inotropic effect of acetylcholine on rat ventricular myocytes.

Acetylcholine (ACh) decreased the contraction of rat ventricular cells within 20 s. ACh (3.1 x 10(-8) M) produced a half-maximal effect and 10(-6) M ACh produced a maximal effect (a 23.8 +/- 5.4% decrease; mean +/- SE, n = 11). During a 3-min exposure to ACh, the inotropic effect faded. Parallel changes were observed in action potential duration: ACh caused an immediate shortening of the action potential, but then the effect faded with time. The changes in action potential duration were the cause of the changes in contraction, because ACh had no effect on contraction when the contractions were triggered by voltage-clamp pulses of constant duration. The changes in action potential duration were the result of the activation of a K+ current (iK,ACh) by ACh. During an exposure to ACh, this current faded as a result of desensitization. iK,ACh was 6.3 times smaller in ventricular than in atrial cells. This may explain why the negative inotropic effect of ACh on atrial cells was greater: 1.0 x 10(-8) M ACh produced a half-maximal effect on atrial cells, and 10(-6) M ACh produced a near maximal effect (a 74.5 +/- 9.5% decrease; n = 4).

Acetylcholine↗

PubMed1993

The relationship between cytosolic free [Ca2+]i concentration and intracellular cAMP of platelet at various stages of experimental atherosclerosis in rabbits.

The relationship between cytosolic free [Ca2+]i and intracellular cAMP of platelet at various stages of experimental atherosclerosis in rabbits was studied. The results showed that the increase of [Ca2+]i was negatively correlated with the level of cAMP in cholesterol-fed rabbits (r = -0.667, P < 0.001) for 2, 4, 8 and 12 weeks. The viscosity of the whole blood at shear rates of 230.4/s and 5.76/s increased only in rabbits fed with cholesterol for 2 weeks, but the plasma viscosity increased in those fed for 2 and 4 weeks.

Animals↗

PubMed1992

Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat.

Despite extensive neurophysiological work carried out to characterize the crossed phrenic phenomenon, relatively little is known about the morphological substrate of this reflex which restores function to a hemidiaphragm paralyzed by spinal cord injury. In the present study WGA-HRP was injected into normal and functionally recovered hemidiaphragm muscle in rats during the crossed phrenic phenomenon. The retrograde transynaptic transport characteristics of WGA-HRP was utilized to delineate the source of the neurons which mediate the crossed phrenic phenomenon. The results indicated that the neurons which drive phrenic motoneurons in spinal hemisected rats during the crossed phrenic phenomenon are located bilaterally in the rostral ventral respiratory group (rVRG) of the medulla. No transneuronal labeling of propriospinal neurons was noted in either normal or spinal-hemisected rats. Thus, propriospinal neurons do not relay respiratory drive to phrenic motoneurons. The neurons of the rVRG project monosynaptically to phrenic motoneurons. The present results suggest that both crossed and uncrossed bulbospinal pathways from the rVRG collateralize to both the left and right phrenic nucleic and functional recovery of a hemidiaphragm paralyzed by ipsilateral spinal cord hemisection is mediated by supraspinal neurons from both sides of the brain stem. These results are important to our complete understanding of the mechanisms which govern motor recovery in mammals following spinal cord injury.

Animals↗

PubMed1992

Retroviral-mediated transfer of the human glucocerebrosidase gene into cultured Gaucher bone marrow.

Gaucher disease, a lysosomal glycolipid storage disorder, results from the genetic deficiency of an acidic glucosidase, glucocerebrosidase (GC). The beneficial effects of allogeneic bone marrow transplantation (BMT) for Gaucher disease suggest that GC gene transduction and the transplantation of autologous hematopoietic stem cells (gene therapy) may similarly alleviate symptoms. We have constructed a retroviral vector, L-GC, produced by a clone of the amphotropic packaging cell line PA317, which transduces the normal human GC cDNA with high efficiency. Whole-marrow mononuclear cells and CD34-enriched cells from a 4-yr-old female with type 3 Gaucher disease were transduced by the L-GC vector and studied in long-term bone marrow culture (LTBMC). Prestimulation of marrow with IL-3 and IL-6, followed by co-cultivation with vector-producing fibroblasts, produced gene transfer into 40-45% of the hematopoietic progenitor cells. The levels of GC expression in progeny cells (primarily mature myelomonocytic) produced by the LTBMC were quantitatively analyzed by Northern blot, Western blot, and glucocerebrosidase enzyme assay. Normal levels of GC RNA, immunoreactive protein, and enzymatic activity were detected throughout the duration of culture. These studies demonstrate that retroviral vectors can efficiently transfer the GC gene into long-lived hematopoietic progenitor cells from the bone marrow of patients with Gaucher disease and express physiologically relevant levels of GC enzyme activity.

Antigens, CD↗

PubMed1992

Antigenic diversity of Rickettsia conorii.

Analysis of seven strains designated as Rickettsia conorii for reactivity with a panel of 12 monoclonal antibodies to surface-protein epitopes of spotted fever group rickettsiae and by Western immunoblotting with standard serotyping sera revealed remarkable antigenic diversity. Rickettsial strains from France, Morocco, Ethiopia, Kenya, South Africa, India, and the USSR differed from one another in reactivity with at least one and as many as five monoclonal antibodies. Simko and Indian strains were similar to one another and differed substantially from other R. conorii strains. All seven strains reacted with three R. conorii-specific monoclonal antibodies. Western immunoblotting demonstrated a major 120-kD protein and a major 135-kD protein in all strains. The principal differences were the presence of a major undenatured 130-kD protein in all strains except Indian and Simko, which had an analogous protein of 124 kD. Immunodominant antigenically related, heat-denatured protein bands of 170 kD (Malish 7 strain), 175 kD (Manuel strain), and 190 kD (Kenya tick typhus, Indian, and Simko strains) were not detected in the M-1 and Moroccan strains. This antigenic diversity is greater than that previously reported for other spotted fever group rickettsial species, suggesting that R. conorrii is an older species than R. rickettsii with a longer period of time for evolutionary divergence.

Animals↗

PubMed1991

Toward gene therapy for Gaucher disease.

We are studying the transfer and expression by retroviral vectors of the human glucocerebrosidase (GC) gene into bone marrow cells as a model of gene therapy for genetic diseases of hematopoietic cells. A simple retroviral vector (G2) was developed that contains a normal human GC cDNA under the control of the Moloney murine leukemia virus long-terminal repeat (LTR) enhancer/promoter. Murine bone marrow was transduced with the G2 vector and maintained in long-term bone marrow culture (LTBMC). Expression of the human GC gene in the transduced murine LTBMC cells exceeded the level of endogenous murine GC mRNA. Murine bone marrow cells were also transduced with G2 and transplanted into irradiated syngeneic recipients. High levels of GC gene transfer and expression were seen in day-12 CFU-S foci, and to a lesser extent in the hematopoietic organs 4 months after gene transfer/bone marrow transplant (BMT). Human bone marrow, from a patient with Gaucher disease, was also used in studies of GC gene transduction. Gene transfer into 35-40% of the Gaucher hematopoietic progenitor cells was achieved, following prestimulation of the marrow with recombinant hematopoietic growth factors. Equal rates of gene transfer were obtained using either total marrow mononuclear cells or progenitor cells enriched 100-fold by immunomagnetic bead separation. GC gene transduction corrected the enzymatic deficiency of the Gaucher marrow. Our results demonstrate the potential utility of retroviral vector-mediated gene transfer for gene therapy of Gaucher disease. Current efforts are aimed at achieving more consistent in vivo GC expression in the murine BMT model and demonstrating transduction of pluripotent human hematopoietic stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Expression of human glucocerebrosidase following retroviral vector-mediated transduction of murine hematopoietic stem cells.

The human glucocerebrosidase (GC) gene has been expressed in the progeny of murine hematopoietic stem cells following transduction of marrow with a retroviral vector (G2) containing the human GC cDNA. Murine marrow was transduced via co-cultivation following prestimulation in the presence or absence of recombinant IL-3 and IL-6. A high rate of gene transfer and expression (95%) was demonstrated in primary day 12 CFU-S foci following bone marrow transplantation (BMT) of G2-transduced marrow into lethally irradiated syngeneic recipient mice. Immunoreactive human GC protein was also documented in the CFU-S foci. Primary recipient mice were examined 4-6 months following BMT. A higher rate of gene transfer (87%) was seen in hematopoietic organs of recipients of prestimulated donor marrow compared with organs from initially unstimulated marrow (25%). A high rate of expression of human GC was also documented in the prestimulated organs (50%) when compared with the unstimulated group (25%). Secondary BMT was performed using marrow from the long-lived primary recipients. The human GC gene was present in 88% of secondary day 12 CFU-S foci examined in the prestimulated group versus 23% in the unstimulated group. Expression of the human GC gene was documented in secondary day 12 CFU-S foci, providing strong evidence of initial hematopoietic stem cell transduction.

Animals↗

PubMed1990

Chronic hypoxia causes morphological alterations in astroglia in the phrenic nucleus of young adult rats.

The present study was carried out to determine if chronic hypoxia in noninjured rats causes morphological alterations in the phrenic nucleus similar to those caused by spinal cord hemisection rostral to the nucleus. Normal rats were subjected to chronic hypoxia in an atmosphere chamber for 48 h. A Bioquant system was used to quantitatively analyze electron micrographs through the phrenic nuclei of chronically hypoxic and normal rats. The results indicated that chronic hypoxia caused astroglial processes to retract from between adjacent dendrites, leading to a significant increase in phrenic dendrodendritic membrane apposition. Unlike spinal cord injury, however, chronic hypoxia did not induce synaptogenesis in the phrenic nucleus. Specifically, the mean length of phrenic dendrodendritic appositions increased significantly from a normal value of 1.42 +/- 0.09 to 1.82 +/- 0.11 microns in hypoxic animals. Moreover, the mean number of dendrodendritic appositions in the normal phrenic nucleus (16.75 +/- 1.11) increased markedly (24.00 +/- 3.54) in the phrenic nucleus of hypoxic rats. There was no significant difference between the mean number of double (17.75 +/- 1.03) or multiple (20.00 +/- 1.47) synapses in normal rats as compared to the mean number of double (18.50 +/- 3.10) or multiple (20.25 +/- 2.84) synapses in the phrenic nucleus of hypoxic animals. From the present results, it is concluded that the rapid synaptogenesis in the phrenic nucleus following spinal cord hemisection is induced by the generalized effects of spinal cord injury and astroglial process retraction does not necessarily lead to synapse formation. The physiological significance of the astroglial movement, however, needs elucidation by additional experiments.

Animals↗