Biomedical subjects
Publications and source records attributed to J Wu.
Lithospermic acid B has been isolated to > 95% purity by high performance liquid chromatography from the aqueous extract of the roots of Salvia miltiorrhiza. When infused at 5.5 mumoles/kg into the post-ischemic rabbit heart, it reduced by 62 +/- 10% (n = 8) the myocardial damage found in the saline control in a rabbit ischemia-reperfusion model.
Conjugated dienes are fingerprint signatures of oxidant damage in cells. We used a radiochemical method based on the Diels-Alder reaction of 14C-labeled tetracyanoethylene with conjugated dienes to delineate the changes of its levels in ischemia-reperfusion in the rat liver. To more directly illustrate the kinetics of diene appearance in hepatocytes, we have applied the same radiochemical assay to rat hepatocytes exposed to xanthine oxidase and hypoxanthine. We observed that the conjugated dienes rose to a maximum under our condition at approximately 10 min, while Trolox--an antioxidant derived from vitamin E found previously to protect rat hepatocytes from oxyradical damage (2)--markedly reduced the formation of conjugated dienes.
The inhibitory effects of a stimulus to the thoracic cord on lumbar segmental reflexes were investigated in the superfused cord of the neonate rat. A single stimulus to the latero-ventral cord surface at T11-T12 evoked fast and slow responses in both L4 ventral roots and inhibited rapid segmental reflexes, both ipsi- and contralaterally. The monosynaptic reflex (MSR) was strongly inhibited and the polysynaptic reflex (PSR) and contralateral fast reflex (CON FAST) were inhibited by 30-40%. The inhibition rose to a maximum 2 sec after the conditioning stimulus, plateaued between 2-20 sec and gradually waned to low levels by 100 sec. The slow segmental responses were not inhibited. Inhibition of the MSR was only elicited ipsilaterally and that of PSR was reduced by about 50% on stimulation of the contralateral thoracic cord; inhibition of CON FAST could be evoked from either side of the cord. Inhibition of the MSR from 2-50 sec was greatly reduced by 5-HT2 receptor antagonists. Ketanserin (1 microM) and ritanserin (1 microM) were equally effective but LY 53857 (1 microM) had a weaker blocking action. Only ketanserin reduced inhibition of the PSR. Prazosin (0.1 microM) did not affect inhibition of the MSR but yohimbine (1 microM) blocked it as effectively as ketanserin. This was probably due to 5-HT2 receptor blockade, since 0.1 microM yohimbine had little blocking action and 1 microM idazoxan none, nor did 0.1 microM clonidine mimic inhibition of the MSR. Inhibition of the MSR and PSR was not reduced by 1 microM naloxone, 1 microM strychnine, 1 microM bicuculline nor 10-30 microM APV. Consistent with the release of 5-HT by descending pathways, the 5-HT uptake blocker, citalopram 0.1 microM and the 5-HT releaser, p-chloroamphetamine 1 microM, depressed segmental reflexes, especially the MSR. 5-Hydroxytryptamine did not have the same depressant action on segmental reflexes as stimulation of the thoracic cord; the slow responses were most affected. Both 8-OH-DPAT (1-3 microM) and dipropyl-5-CT (1 microM) preferentially depressed the MSR. Neither spiroxatrine (0.1 microM) nor methysergide (5-10 nM) altered inhibition of the MSR. The concentration of ketanserin required to reduce sub-maximal inhibition by 50% was estimated using 2 concentrations of antagonist. The pIC50, estimated for the blockade by ketanserin of inhibition 20-50 sec after a conditioning stimulus, was 7.3-7.5. It is concluded that inhibition of the MSR and PSR does not involve mediation by glycine, GABAA nor NMDA receptors, nor release of enkephalins nor noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)
Theoretical models for calculating the on-axis acoustic streaming velocity generated by a continuous-wave (CW) and pulsed finite amplitude ultrasonic beams have been developed. Using these models, the on-axis streaming velocity is calculated for unfocused linear plane wave ultrasonic beams, focused linear Gaussian beams, and unfocused ultrasonic tonebursts of finite amplitude. The numerical results show that the on-axis streaming velocity is significantly enhanced by the nonlinear harmonic generation of finite-amplitude ultrasonic tonebursts.
The feasibility of using antigenically disguised skin xenotransplants to cover extensive burns for a suitable time lag without administering immunosuppressive drugs was tested experimentally. Pieces of human skin that had been preincubated for 3 h at 37 degrees C with either mouse anti-human beta 2-microglobulin monoclonal antibody (beta 2m-McAb) or PBS (controls) were grafted onto the backs of immunologically competent Swiss mice, and the time required for their rejection or substitution by normal autogenous skin was determined. Thus, it was found that the beta 2m-McAb-pretreated xenografts had a significantly longer mean survival time than the control grafts. An even longer skin xenograft MST was obtained when beta 2m-McAb was repeatedly injected, at weekly intervals, just beneath the transplants. Parallel immunohistochemical studies showed that the beta 2m-McAb entered the grafts and was bound to its targets both in epidermis and dermis. Moreover, a small amount of beta 2m-McAb administered at the outset significantly hindered the reactive proliferation of primed mouse spleen cells cultured in the presence of human epidermal cells. Finally, neither toxic effects nor a weakening of immune competence were elicited by repeated intraperitoneal injections of beta 2m-McAb. Therefore, it seems expedient to propose the use of beta 2m-McAb to delay the rejection of skin xenografts as this antibody harmlessly prevents, wholly or in part, the activation of the recipients' lymphocytes. This would positively aid any patient urgently needing xenograft cover of extensive burns.
A comparative evaluation was made of the conventional spectrophotometric procedure and three published high performance liquid chromatographic (HPLC) procedures for the determination of malondialdehyde (MDA) as the thiobarbituric acid (TBA) derivative when applied to liver, fish meal, serum, and urine. Except for urine, spectrophotometric analysis overestimated MDA content. Purification of the TBA-MDA complex obtained from liver and fish meal on reverse phase cartridges was found to entail a loss of complex bound to residual peptides in the trichloracetic acid (TCA) extract. Mincing as opposed to homogenizing liver samples led to a doubling of values for MDA content. Hexanal was a major TBA reactant, in addition to MDA, in all the samples. Acid hydrolysis and heat were necessary for the release of MDA bound to the amino groups of proteins and other amino compounds. Methods for free MDA have limited application to biological materials except short term in vitro preparations such as peroxidizing microsomes, in which free MDA accumulates. On the basis of these and other observations, a modified HPLC procedure for the determination of MDA as the TBA-MDA complex is proposed.
Previous studies have indicated that schizophrenic patients have olfactory deficits. The question as to whether olfactory deficits are due to chronic effects of medication has not been addressed. This is the first paper to report that never-medicated schizophrenic patients also have olfactory deficits. Twenty four normal subjects and twenty unmedicated schizophrenic patients were examined with two tests of olfactory function: the University of Pennsylvania Smell Identification Test (UPSIT) and a match-to-sample olfactory memory test. Results indicated that schizophrenics did poorly on both the UPSIT and the olfactory match-to-sample memory test relative to sex and age-matched controls. ANCOVA showed that the deficit in performance on the olfactory match-to-sample test was still present even when the variance due to the UPSIT was taken out of the analysis. Deficits in olfactory identification and olfactory memory are consistent with the concept that schizophrenics have dysfunctional limbic systems.
Rapamycin (RAPA) is a strong immunosuppressant and is able to prevent allograft rejection in animal models. We have demonstrated that RAPA could strongly inhibit in vitro immunoglobulin (Ig) production by human lymphocytes. The present study investigated the long-term in vivo effect of RAPA on humoral and cellular immune responses, and the effect of RAPA on accelerated rejection. It was shown that RAPA strongly inhibited antigen (Ag) specific antibody (AB) production (i.e. cytotoxic Ab to donor lymphocytes and Ab to tetanus toxoid) during the period of drug administration. The accelerated rejection of cardiac allografts in presensitized animals was alleviated by RAPA administration. These results suggest the potential application of RAPA in treatment of presensitized candidates for organ transplantation. A little more than two months after the drug withdrawal, the rats were basically competent in Ab response to further Ag challenges. When tested 4 months after the RAPA-treatment, the rats showed uncompromised cardiac allograft rejection, and the cellular immune response in vitro according to mixed lymphocyte reaction (MLR) and mitogen-stimulated proliferation were not hampered. Such results suggest that the immune system can return to normal status without sequelae after discontinuation of RAPA.
This study examines the circadian variation of aortic blood flow velocity, acceleration, and distance of stroke, assessed every 4 hours for 24 hours by Doppler ultrasound in 88 human newborns. Groups 1 and 2 involve neonates with a negative family history of high blood pressure or myocardial infarction monitored during the first day or between the second and tenth days postpartum, respectively; groups 3 to 6 are 2- to 10-day-old babies with a positive family history of high blood pressure (group 3) or of myocardial infarction (group 4) or with a negative family history and intrauterine growth retardation (group 5) or born prematurely (group 6). Cosinor analyses demonstrate a circadian rhythm for all variables in groups 2 and 5 (p < 0.001), with overall high values occurring around 17:40. In group 2, a circadian rhythm remains demonstrable in boys and girls considered separately, and mostly so in arbitrary subgroups of different sizes, with only slight differences in rhythm parameters among them. These results show that, with relatively small samples, reproducible circadian rhythms of cardiac function can be detected shortly after birth in neonates with a negative family history of high blood pressure or myocardial infarction.
We describe a novel Triton-disrupted mammalian cell system wherein the pathways for activation of mitogen-activated protein (MAP) kinases (MAPKs) are capable of direct biochemical manipulation in vitro. MAPKs p42mapk and p44mapk are activated in signal transduction cascade(s) initiated by occupancy of plasma membrane receptors for peptide growth factors, hormones, and neurotransmitters. One likely activation pathway for MAPKs consists of sequential activations of c-ras, c-raf-1, and a protein-tyrosine/threonine kinase, MAP kinase kinase. Triton-disrupted cells retained capacity for activation of the pathway by both peptide growth factors and by addition of GTP-loaded p21 rasVal12. Incubation of disrupted cells with an antibody that neutralized the function of c-ras (Y13-259) abolished receptor-mediated stimulation of MAPK as did acute addition of 200 microM azatyrosine. Activation of the pathway was reconstituted in a cell-free system using high-speed supernatants generated from Triton-disrupted cells together with purified plasma membranes from parental cells and as a heterogeneous system using purified plasma membranes from v-ras-transformed cells. These systems will allow biochemical dissection in vitro of the interaction(s) between c-ras and the MAPK pathway in mammalian cells.
OBJECTIVE: The aim was to determine if lactosylphenyl Trolox could improve myocardial resistance to ischaemia and reperfusion. Lactosylphenyl Trolox is derived by coupling p-aminophenyl-beta-D-lactopyranoside to Trolox. Trolox, a polar analogue of vitamin E, has been found to protect human cardiomyocytes against oxyradicals and to reduce myocardial damage by 66% in a canine ischaemia-reperfusion model. METHODS: New Zealand white rabbits (weighing approximately 3.5 kg) were subjected to 1 h ischaemia by ligation of the main branch of the anterior ventricular coronary artery. Approximately 1-2 min before release of ischaemia, a 30 ml bolus of saline (placebo control) or saline containing lactosylphenyl Trolox was injected into the right external jugular vein, followed by 3 h reperfusion. The area at risk was identified by staining with Evans Blue. Area of necrosis was indicated by tetrazolium red staining, confirmed by histopathology and quantified by planimetry. RESULTS: The control group (n = 6) had 46.6(SD 10.0)% necrosis of the area at risk but the lactosylphenyl Trolox treated groups (n = 6 per group) had reduced necrosis: 34.0(6.5)%, 17.4(8.2)%, and 6.9(3.6)% at doses of 2.5, 5.0, and 10 mumol.kg-1, respectively (each with p < 0.05 v control value). These translated to 48.6(14.0)%, 62.7(17.6)%, and 85.3(7.8)% myocardial salvage, respectively. In contrast, the salvages achieved with 2.5 and 10 mumol.kg-1 of Trolox were 31.0(11.0)% and 62.1(18.9)% respectively (both p < 0.05 v lactosylphenyl Trolox). CONCLUSIONS: Lactosylphenyl Trolox reduces myocardial infarct size more effectively than Trolox in a rabbit model of ischaemia-reperfusion.
In canine experimental models of femoral arterial and venous thrombosis, the feasibility of thrombus imaging with 99Tcm-labelled monoclonal antibody SZ-51 to an alpha-granule membrane protein (GMP-140) on the surface of activated platelets was investigated. Both the arterial and venous thrombi were clearly discernible at 2 to 8 h after injection of 99Tcm-SZ-51. The suitable imaging time was 4 to 6 h. These results were confirmed by ex vivo images of thrombosed arteries and veins followed by killing the dogs at 8 h postinjection of 99Tcm-SZ-51. Quantitative imaging analysis showed that the ratios between the thrombus and the opposite vessel were increased strikingly over time. The ratios of thrombus to blood or surrounding muscle were 57.09 +/- 12.24 and 177.46 +/- 9.00 for arterial thrombi and 6.43 +/- 4.70 and 83.22 +/- 68.98 for venous thrombi at the time of death. Radiolabelled antibody was cleared from the blood with T1/2 alpha of 16.25 +/- 13.39 min and T1/2 beta of 7.28 +/- 1.15 h. These results show that it is feasible to detect noninvasively thrombus formation in vivo using 99Tcm-labelled monoclonal antibody SZ-51.
Rapamycin was examined for its effects on reversal of ongoing rejection of heart, kidney, and pancreas allografts and on suppression of accelerated heart allograft rejection in the rat. A 14-day continuous intravenous infusion of RAPA by an osmotic pump at 0.02, 0.08, and 0.8 mg/kg/day to WFu recipients, starting 4 days postoperation, significantly protected the BUF heart allografts with a mean survival time (MST) +/- 1 SD of 33.2 +/- 19.8 (p < 0.001), 48.2 +/- 14.8 (p < 0.001), and 107.0 +/- 86.3 (p < 0.001) days, respectively, as compared with 7.2 +/- 0.8 days in vehicle-treated controls. Combination of low dose RAPA (0.02 mg/kg or 0.08 mg/kg) and low dose CsA (2 mg/kg) achieved significantly longer cardiac allograft survival than RAPA or CsA alone. RAPA's effect in reversing ongoing rejection of renal and pancreatic allografts was also significant. The BUF kidney and pancreas in WFu recipients treated with a 14-day course of RAPA (0.8 mg/kg/day starting 4 days postoperation) had an MST of 44.7 +/- 15.9 (p < 0.001) and 46.4 +/- 12.5 (p < 0.001), while in vehicle-treated controls, the grafts were rejected within 10 days. RAPA could also suppress accelerated cardiac allograft rejection. Hyperimmunized WFu recipients were treated with two 14-day courses of continuous i.v. RAPA at 0.8 mg/kg/day before and after BUF heart allografting. Significantly longer survival of the grafts (25.5 +/- 3.7 days, p < 0.001) was achieved compared with that of the vehicle-treated controls (3.8 +/- 1.0 days). One-course RAPA treatment before or after heart transplantation was considerably less effective. RAPA was also shown to prevent the increase of serum IgG levels and to inhibit the production of specific cytotoxic Ab in the rat receiving repetitive immunizations. Such effects presumably contribute to the inhibition of the accelerated rejection. The results of this study suggest that RAPA is potentially useful in treatment of ongoing as well as accelerated allograft rejection.
Rapamycin (RAPA) is a potent immunosuppressant. In this study we investigated the effect of RAPA on T cell proliferation triggered by various stimuli in an in vitro human model. The proliferation of T cells stimulated via an alternative pathway using phorbol myristate acetate (PMA) and anti-CD28 antibody (alpha CD28) in the absence of antigen-presenting cells (APC) was strongly inhibited by RAPA. T cell proliferation provoked via a combination of CD3/TCR and CD28 pathways using anti-CD3 antibody (alpha CD3) plus alpha CD28 was also inhibited by RAPA in the presence of APC. The mitogen (phytohaemagglutinin (PHA) or alpha CD3)-induced up-regulation of expression of the IL-2 receptor alpha chain (IL-2R alpha) and the IL-4 receptor (IL-4R) was sensitive to RAPA. This suggests that RAPA's interference with the IL-2 and IL-4 autocrine loops during T cell activation might contribute to RAPA's overall immunosuppressive effect. We have further demonstrated in a two-stage culture system that RAPA strongly inhibited IL-4-stimulated proliferation of T cells, the latter being either pretreated with alpha CD3 in the presence of APC, or with PMA plus alpha CD28 in the absence of APC. The result suggests that the Ca++ influx during the pretreatment is not obligatory for T cells to achieve IL-4 responsiveness. The results also indicate that RAPA's antiproliferative effect on IL-4-stimulated T cells is not contingent on the various mechanisms of cell priming. Therefore, RAPA's major target is probably at the second stage after the priming. Our study has extended current knowledge about the effect of RAPA on human T cells.
The plasmid-encoded arsenical resistance (ars) operon produces resistance to trivalent and pentavalent salts of arsenic and antimony. The first gene in the operon, arsR, was previously shown to encode a repressor protein. A newly identified gene, arsD, is shown here to encode a regulatory protein, the ArsD protein. The gene was identified by construction of an in-frame fusion between the C-terminally truncated arsD gene and the coding region for the mature form of beta-lactamase (blaM). The native arsD gene product was overexpressed and radioactively labelled as a 13 kDa polypeptide. A frameshift mutation within the arsD gene resulted in elevated levels of expression of downstream ars genes. Co-expression of a wild-type arsD gene in trans with the operon containing the mutated arsD gene reduced expression of the downstream genes to wild-type levels. The presence of the arsD gene had no effect on the basal level of operon expression set by the arsR gene product, and the repression produced by the arsD gene product was not affected by inducers of the operon. The results indicate that the ArsD protein is an inducer-independent trans-acting regulatory protein.
Myocardial ischemia in vivo is associated with dramatic electrophysiologic alterations that occur within minutes of cessation of coronary flow and are rapidly reversible with reperfusion. This suggests that subtle and reversible biochemical alterations within or near the sarcolemma may contribute to the electrophysiologic derangements. Our studies have concentrated on two amphipathic metabolites, long-chain acylcarnitines and lysophosphatidylcholine (LPC), which have been shown to increase rapidly in ischemic tissue in vivo and to elicit electrophysiologic derangements in normoxic tissue in vitro. Incorporation of these amphiphiles into the sarcolemma at concentrations of 1 to 2 mole%, elicits profound electrophysiologic derangements analogous to those observed in ischemic myocardium in vivo. The pathophysiological effects of the accumulation of these amphiphiles are thought to be mediated by alterations in the biophysical properties of the sarcolemmal membrane, although there is a possibility of a direct effect upon ion channels. Inhibition of carnitine acyltransferase I (CAT-I) in the ischemic cat heart was found to prevent the increase in long-chain acylcarnitines and LPC and to significantly reduce the incidence of malignant arrhythmias including ventricular tachycardia and fibrillation. This review focuses on the electrophysiologic derangements that are observed during early ischemia and presents data supporting the concept that accumulation of these amphiphiles within the sarcolemma contributes to these changes. The potential contribution of these amphiphiles to the increases in extracellular potassium and intracellular calcium are examined. Finally, recent data pertaining to the accumulation of long-chain acylcarnitines on cell-to-cell uncoupling are presented. In addition to the events reviewed here, there are many other alterations that occur during early myocardial ischemia, but the results from multiple studies over the past two decades indicate that the accumulation of these amphiphiles contributes importantly to arrhythmogenesis and that development of specific inhibitors of CAT-I or phospholipase A2 may be a promising therapeutic strategy to attenuate the incidence of lethal arrhythmias associated with ischemic heart disease in man.
The human cytomegalovirus major immediate-early gene encodes several protein isoforms which autoregulate the major immediate-early promoter (MIEP). One of these isoforms, the IE86 protein (UL122, IE2), is a DNA-binding protein that represses the MIEP through its cognate recognition sequence (designated the cis repression signal [crs]) located between the TATA box and the initiation site of transcription. Purified recombinant IE86 protein was shown to repress MIEP transcription in vitro, in a cis-acting mediated pathway, with nuclear extracts from HeLa S3, U373-MG, and primary human foreskin fibroblast cells. Repression of the MIEP by IE86 was shown by two criteria to be dependent on the direct interaction of IE86 with the crs element. Core promoter constructs containing essentially the MIEP TATA box and crs element were also specifically repressed by IE86 but not by a mutant IE86 protein, indicating the general transcription machinery as the target for IE86 repression. Kinetic and template commitment experiments demonstrated that IE86 affects preinitiation complex formation but not the rate of reinitiation. Sarkosyl inhibition experiments further revealed that IE86 was unable to effect repression by either disassembling or preventing the elongation of a preexisting transcription complex. Further, the ability of IE86 to interact with the DNA-binding subunit of TFIID was shown not to be required for repression. These functional protein-DNA and protein-protein interaction experiments demonstrate that IE86 specifically interferes with the assembly of RNA polymerase II preinitiation complexes. The biological significance of these results and the precise mechanism by which IE86 represses transcription are discussed.
Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.