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Biomedical subjects

D Song

Publications and source records attributed to D Song.

At least 55 records · Page 3Linked to original sources
PubMed1999

Effects of hyperosmolarity and ion substitutions on amino acid efflux from the ischemic rat cerebral cortex.

The contributions of sodium and chloride ions and of osmotic stresses to the ischemia-evoked efflux of excitotoxic and other amino acids were explored using a rat four vessel occlusion model. Replacement of Na+ with choline or N-methyl-D-glucamine (NMDG) and of Cl- with sulfate or gluconate was used to evaluate the contribution that these ions make to amino acid efflux. The contribution of ischemia-evoked swelling to amino acid release was studied by applying mannitol or sucrose to minimize the cell volume increases and the compensatory regulatory volume decrease evoked efflux of amino acids. Aliquots of artificial cerebrospinal fluid (aCSF), appropriately adjusted for ion replacement or 150 mM mannitol or sucrose, were pipetted into cortical cups 35 min prior to ischemia and perfusate samples were obtained prior to, during and following ischemia (20 min) and reperfusion (40 min). Replacement of Na+ by NMDG depressed basal (normoxic) efflux of most amino acids, with choline substitution having little effect. During ischemia NMDG substitution increased glutamate and GABA efflux and choline enhanced the release of aspartate, glutamate, GABA and taurine. A reduction in extracellular Na+ would facilitate reversal of Na+-dependent transporters with extrusion of amino acids. Another possible explanation for the elevated release is that the absence of Na+ would inhibit the Ca2+/Na+ counter transport system, with a deleterious accumulation of intracellular Ca2+. Chloride replacement with sulfate or gluconate enhanced the efflux of aspartate, glutamate, GABA and taurine during ischemia. Removal of Cl- would depolarize cells, and block the Cl--dependent action of inhibitory amino acid transmitters, with both actions enhancing the ischemic injury and, consequently, amino acid release. Exposure to hyperosmotic mannitol (150 mM) aCSF enhanced ischemia-evoked release of some amino acids (taurine, GABA) and decreased that of aspartate and phosphoethanolamine. Sucrose aCSF enhanced the ischemia-evoked release of most amino acids. A potential explanation for these observations is that both agents may be able to rapidly penetrate the plasma membranes of ischemic neurons, actually contributing to the release of other osmolytes. The unanticipated nature of many of the observations made during these manipulations of the aCSF serves to accentuate the complex nature of the mechanisms responsible for the ischemia-evoked amino acid efflux into the interstitial spaces.

Animals↗
PubMed1999

Regional expression of heat shock protein 72 mRNA following mild and severe hypoxia in neonatal piglet brain.

The present study examined the effect of hypoxia on expression of 72-kDa heat shock protein (hsp72) mRNA in the newborn brain. The studies were carried out in anesthetized and mechanically ventilated newborn piglets, age 3-5 days. Hypoxic insult was induced by decreasing the fraction of inspired oxygen (FiO2) from 21% to 6% or 10% for 1 h. Oxygen pressure in the microvasculature of the cortex (cortical pO2) was measured by oxygen dependent quenching of the phosphorescence of phosphor dissolved in blood. Following the two hours of normoxic recovery, regional expression of the 72-kDa heat shock protein (hsp72) mRNA was determined using in situ hybridization and autoradiography. Two grades of hypoxia were studied. Mild hypoxia (cortical pO2 = 10-30 mm Hg) induced the expression of hsp72 mRNA predominantly in the subcortical white matter. In individual animals of this group, the extent of expression varied from isolated regions to widespread involvement of the white matter. Severe hypoxia (cortical pO2 = 3-10 mm Hg) induced the expression of hsp72 mRNA in both white and gray matter regions, with strong expression occurring in the cerebral cortex of individual animals. The present results indicate that immature white matter is more sensitive than gray matter to the hypoxia induced expression of hsp72 mRNA. Further, increased expression of hsp72 mRNA may be an indicator of a pathologic degree of hypoxic stress, and the observed increase may indicate that in the newborn brain the immature white matter is particularly sensitive to injury by hypoxia-ischemia and reperfusion.

Animals↗
PubMed1999

Increased nitric oxide synthase expression in aorta of cirrhotic rats.

The characteristic cardiovascular changes in liver cirrhosis are vasodilatation and increased cardiac output. Augmented activity of the vasorelaxant factor, nitric oxide (NO), stimulated by cytokines, have been suggested to play a role in the pathogenesis, but previous studies show conflicting results. We therefore aimed to evaluate the entire pathway from cytokines to the final metabolites, nitrate/nitrite. The levels of serum Tumor Necrosis Factor-alpha (TNFalpha) and nitrate/nitrite (NOx) were measured, and aorta content of inducible (iNOS) and endothelial nitric oxide synthase (eNOS) mRNA and protein were determined by reverse-transcription polymerase chain reaction and Western blotting in rats with cirrhosis due to chronic bile duct ligation and sham-operated controls. Compared to control rats, serum TNFalpha levels were significantly elevated in cirrhotic rats (48.4+/-21.1 vs 16.8+/-9.0 pg/ml, p<0.01); iNOS mRNA was detectable whereas it was absent in controls, and eNOS mRNA levels was significantly higher in aortae of cirrhotic rats. Aortic eNOS protein content was significantly higher in cirrhotic rats, but iNOS protein was undetectable by Western blotting in both groups. Serum NOx concentrations in the cirrhotic group were significantly higher than those in controls (3.5+/-1.0 vs 2.3+/-0.5 microM, p<0.01). These results suggest that NO activity in cirrhosis is increased, and is predominantly due to eNOS since the detectable iNOS mRNA does not seem to be expressed as protein. The increased NOS activity in the arterial system may play a role in the systemic hemodynamic changes occurring in cirrhosis.

Animals↗
PubMed1999

Role of nitric oxide in rat coronary flow regulation during respiratory and metabolic acidosis.

A rat Langendorff heart preparation, perfused at constant pressure, was used to evaluate the role of nitric oxide in the increases in coronary vessel flow during hypercapnic and metabolic acidosis. Prior administration of the nitric oxide synthase (NOS) inhibitor N6-nitro-L-arginine methyl ester (100 microM) significantly reduced the basal, resting, rate of coronary flow but did not attenuate the increases in flow during brief (2-min) periods of perfusion with acidotic solutions. These results suggest that nitric oxide is not a significant contributor to rat heart coronary flow regulation during respiratory or metabolic acidosis.

Acidosis↗
PubMed1999

Further evidence for the role of adenosine in hypercapnia/acidosis-evoked coronary flow regulation.

Experiments were performed on isolated, nonworking rat hearts perfused at constant pressure according to the Langendorff technique to evaluate the role of adenosine in hypercapnia-evoked coronary vasodilation. Hypercapnia/acidosis resulted in increases in heart rate and coronary flow rates in conjunction with a decrease in ventricular contractile tensions. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, 10 microM) reduced the heart rate and enhanced CO2-evoked increases in coronary vascular flow. 5-Iodotubercidin (1 microM), an inhibitor of adenosine kinase, caused a reduction in heart rate and enhanced coronary flow rates during hypercapnic perfusion. Adenosine deaminase (1 U/ml) significantly attenuated CO2-evoked increases in coronary vascular flow. These results extend those of previous investigations implicating adenosine in the regulation of coronary flow during conditions of respiratory or metabolic acidosis.

Acidosis↗
PubMed1999

Remifentanil as an adjuvant during desflurane anesthesia facilitates early recovery after ambulatory surgery.

STUDY OBJECTIVE: To investigate the effect of using a remifentanil infusion during desflurane anesthesia on the early recovery profile and side effects. DESIGN: Randomized, single-blind study. SETTING: University-based ambulatory surgery unit. PATIENTS: 46 healthy, ASA physical status I and II women undergoing outpatient laparoscopic tubal ligation procedures. INTERVENTIONS: After premedication with midazolam 2 mg intravenously (IV), anesthesia was induced with propofol 2 mg.kg-1 i.v. and remifentanil 1 microgram.kg-1 i.v. Following tracheal intubation, anesthesia was maintained with desflurane 2% and nitrous oxide (N2O) 65% in both groups. During the maintenance period, hemodynamic stability was maintained using either a variable inspired concentration of desflurane, 2% to 8% (Control group), or a variable-rate infusion of remifentanil 0.05 to 0.2 microgram.kg-1.min-1 i.v. (Remi group). Ketorolac 30 mg i.v. and local anesthetic infiltration at the surgical portals were administered for preventive analgesia prior to skin closure. MEASUREMENTS AND MAIN RESULTS: Emergence times and times to achieving an Aldrete score of 10 (i.e., fast-tracking eligibility) were determined. Postoperative nausea and vomiting (PONV), as well as the need for analgesic and antiemetic rescue medications, were noted during the 24-hour follow-up period. A structural questionnaire was used to assess intraoperative recall. Compared to the Control group, the Remi group had shorter emergence times and reduced times to achieving an Aldrete score of 10. There were no differences between the two groups with respect to the incidence of PONV and the requirements for postoperative analgesic and antiemetic drugs. None of the patients experienced intraoperative recall. CONCLUSIONS: The adjunctive use of a remifentanil infusion (0.07 +/- 0.03 microgram.kg-1.min-1) during desflurane-N2O anesthesia facilitated early recovery without increasing PONV, pain, or the need for rescue medication after laparoscopic surgery.

Ambulatory Surgical Procedures↗
PubMed1999

Effects of anion channel blockers on hyposmotically induced amino acid release from the in vivo rat cerebral cortex.

A cortical cup model with continuous perfusion of artificial cerebrospinal fluid (containing 134 mM NaCl) was used to investigate the effects of anion channel blockers on the hyposmotically-induced release of amino acids from the in vivo rat cerebral cortex. The hyposmotic stimulus (25 mM NaCl) evoked a release of taurine, glutamate, aspartate, glycine, phosphoethanolamine and GABA. Topically applied anion channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (1 mM); 4-acetamido-4'-isothiocyanatostilbene-2,2-disulfonic acid (2 mM); 5-nitro-2-(3-phenylpropylamino) benzoic acid (350 microM); niflumic acid (500 microM); tamoxifen (20 microM) and arachidonic acid (0.5 microM) all significantly reduced the hyposmotically-induced release of taurine. The releases of glutamate, aspartate, glycine, phosphoethanolamine and GABA were variably susceptible to inhibition by these compounds. These results demonstrate that osmoregulatory processes in cortical cells, in vivo, involve amino acids, with taurine playing a dominant role. The efflux of taurine and, to a lesser extent, the other amino acids may be mediated by anion channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Activation of cPLA2, PKC, and ERKs in the rat cerebral cortex during ischemia/reperfusion.

Release of the excitotoxic amino acid, glutamate, into the extracellular space during ischemia/reperfusion contributes to neuronal injury and death. To gain insights into the signal transduction pathways involved in glutamate release we examined the time course of changes in enzyme levels and activities of cPLA2, PKC and ERKs in the rat cerebral cortex after four vessel (4VO) ischemia followed by reperfusion. Measurement both by enzymatic assay and Western blot analysis showed significant increases in the activity and protein levels of cPLA2 during 10-20 min of ischemia. Activity remained elevated at 10 min and 20 min of reperfusion, whereas cPLA levels had returned to base line levels after 20 min of reperfusion. PKC activity increased significantly in the particulate, but not in the cytosolic, fractions both during ischemia and reperfusion. Increases in PKCgamma levels were recorded in the particulate fraction during ischemia and reperfusion, and in the cytosolic fraction during ischemia. Western blot analysis with a phosphospecific antibody for characterization of MAPK (ERKs) activation revealed significantly increased phosphorylation of ERK1 and ERK2 in the particulate fraction, of ERK2 in the cytosolic fraction, during ischemia and of both enzymes in the particulate and cytosolic fractions after 10 min of reperfusion. The relevance of the results to glutamate release is discussed.

Animals↗
PubMed1999

Random transposition by Tn916 in Desulfitobacterium dehalogenans allows for isolation and characterization of halorespiration-deficient mutants.

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.

Bacteria, Anaerobic↗
PubMed1998

5-(N-Ethyl-N-isopropyl)-amiloride inhibits amino acid release from the ischemic rat cerebral cortex: role of Na+-H+ exchange.

The effect of the selective Na+/H+ antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on amino acid release from the ischemic/reperfused rat cerebral cortex was investigated using a cortical cup technique. EIPA (25 microM in artificial cerebrospinal fluid), applied topically, inhibited the ischemia-reperfusion evoked efflux of aspartate, glutamate, gamma-aminobutyric acid, taurine and phosphoethanolamine. Reductions in the ischemia-evoked releases of these amino acids suggest that ischemia precipitates acidosis, Na+/H+ exchange and cell swelling with amino acid release as the cells mount a regulatory volume decrease response. EIPA, by blocking Na+/H+ exchange, would reduce cell swelling and the resulting amino acid release.

Amiloride↗
PubMed1998

The role of adenosine in rat coronary flow regulation during respiratory and metabolic acidosis.

The role of adenosine in rat coronary flow regulation during acidosis was evaluated in isolated, perfused, Langendorff rat heart preparations exposed to brief periods of hypercapnic or metabolic acidosis. Acidosis resulted in increases in coronary flow rate, in conjunction with decreases in ventricular contractile tensions. Heart rates were non-significantly increased. Two non-selective adenosine antagonists, caffeine and 8-phenyltheophylline, markedly attenuated the increases in coronary flow during hypercapnic acidosis without affecting the decline in contractile tension or the heart rate. ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]triazin-5-ylami no]ethyl)phenol), a selective adenosine A2A receptor antagonist, also blocked hypercapnic acidosis-evoked coronary flow rate increases. The adenosine A1 selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, did not affect flow rate increases during hypercapnic acidosis. SCH 58261 (5-amino-7-(2-phenyl ethyl)-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine, a selective adenosine A2A receptor antagonist, blocked the increases in coronary flow rate evoked by metabolic acidosis. An adenosine transport inhibitor, dipyridamole, doubled coronary flow rates during hypercapnic acidosis. When taken in conjunction with previous reports that acidosis enhances adenosine release from cardiac preparations, these results suggest that adenosine is a significant contributor to acidosis-evoked increases in coronary flow.

Acidosis↗
PubMed1998

DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression.

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between -14.6 kb and -32 kb 5' to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (-14.6 to -16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen in vivo. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.

3T3 Cells↗
PubMed1998

Mechanisms of amino acid release from the isolated anoxic/reperfused rat heart.

Loss of amino acids into the coronary artery perfusate, which is exacerbated during anoxic stress, may have important implications for the ability of hearts subjected to ischemia or anoxia to recover function during reoxygenation. This work investigates the mechanisms underlying the amino acid efflux. Rat Langendorff heart preparations were used to study amino acid loss into coronary artery perfusates during anoxia or anoxia/reoxygenation sequences. Coronary flow rates, heart rates and intra-aortic pressures were recorded. Changes in myocardial amino acid concentrations were equated with amino acid levels in collected anoxic perfusate. With the exception of taurine, the differences in amino acid levels between normoxic and anoxic hearts were smaller than the amounts lost into the coronary perfusates, indicating ongoing replenishment of most amino acids during the anoxic episode. Fifteen-minute periods of exposure to low oxygen levels (P02 18-20 mmHg) resulted in large percentage increases in perfusate amino acid levels which returned slowly towards control levels upon reoxygenation. Anion channel blockers, anthracene-9-carboxylic acid, furosemide, and 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid (SITS), depressed anoxia-elicited increases in amino acid release. Phospholipase inhibition with quinacrine, 4-bromophenacyl bromide and 7,7-dimethyl-eicosadenoic acid (DEDA) depressed the anoxia-evoked release of amino acids. Combined applications of SITS and DEDA exhibited additive effects, virtually abolishing anoxia-evoked release of all the amino acids. The protein kinase C inhibitor, chelerythrine chloride, and the protein tyrosine kinase inhibitors, genistein and lavendustin A, inhibited anoxia-evoked amino acid release. Polyunsaturated fatty acids, arachidonic and linoleic, reduced anoxia-evoked amino acid release whereas monosaturated (oleic) and saturated (stearic) acids were ineffective. The glutamate transport inhibitor, dihydrokainate, depressed anoxia-evoked glutamate and aspartate release. These results suggest that at least three possible mechanisms for the anoxia-evoked amino acid efflux including (a) diffusional release through volume activated anion channels, (b) leakage across myocyte plasma membranes as a consequence of phospholipase activation and (c) reversal of Na+ dependent high-affinity transporters.

Amino Acids↗
PubMed1998

Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria.

Methionine synthase catalyzes the remethylation of homocysteine to methionine via a reaction in which methylcobalamin serves as an intermediate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl donor. Patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a cDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted molecular mass of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. The authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affected siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1998

Interaction of Oct-1 and automodification domain of poly(ADP-ribose) synthetase.

We isolated several clones from a matchmaker two-hybrid system human lymphocyte cDNA library using an automodification domain of poly(ADP-ribose) synthetase (PARS) as a probe. A DNA sequence (approximately 1 kbp) of the clone was identical to part of the Oct-1 DNA sequence. We then constructed either a His-tagged or GST fusion protein of the inserted cDNA from the clone and the fusion protein was shown to interact with PARS by far-Western blot analysis and co-precipitation with affinity resin. Furthermore, the His-tagged Oct-1/POU-homeo fusion protein interacted weakly with the octamer motif of the DRa promoter and the addition of PARS fusion protein greatly increased the DNA binding activity. These results suggest that PARS interacts with Oct-1 and stabilizes the binding of Oct-1 to the octamer motif.

Base Sequence↗