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J Y Chan

Publications and source records attributed to J Y Chan.

At least 109 records · Page 6Linked to original sources
PubMed1992

Identification of a specific inhibitor for DNA ligase I in human cells.

A protein inhibitor for human DNA ligase I has recently been identified. It was copurified with a fraction of the enzymes from HeLa cells through several steps of chromatography. The inhibitor was first identified by the absence of ligation activity of the associated enzyme, while it retained the ability to form the ligase-[32P]AMP adducts. The inhibitor was eluted as a single peak at approximately 0.25-0.30 M NaCl from a Mono S column. It inhibited the ligation of both double-stranded and single-stranded breaks by purified DNA ligase I but not by T4 DNA ligase and DNA ligase II. Subsequent gel-filtration chromatography indicated that this inhibitor, with a molecular mass of 55-75 kDa, could form a complex with DNA ligase I and inhibited the DNA ligation activity. Rechromatography of the ligase I-inhibitor complex in high-salt conditions resulted in the dissociation of the complex and the restoration of enzyme activity, indicating that the physical interaction of inhibitor with DNA ligase I is one of the mechanisms of inhibition. These data indicate that this protein inhibitor for DNA ligase I may play a specific role in regulating DNA ligation during replication, repair, or recombination.

Chromatography, Affinity↗
PubMed1992

Dual mode of inhibition of purified DNA ligase I from human cells by 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (F-ara-A) is an analogue of adenosine and deoxyadenosine with potent anti-tumor activity. The mechanism of action for this compound has been elucidated as the inhibition of DNA and RNA synthesis, induction of DNA fragmentation, and genetic damage. This study demonstrated that DNA ligase I, an enzyme involved in DNA replication, is a target for the drug action. F-ara-adenine triphosphate (F-ara-ATP) at 80 microM inhibited the activity of DNA ligase I by more than 90%. In contrast, eight other related nucleoside analogues showed no effect on the enzyme activity at 200 microM. F-ara-ATP inhibited DNA ligation in two distinct ways. First, F-ara-ATP directly interacted with DNA ligase I and inhibited the formation of the ligase-AMP complex. This inhibition could not be reversed when free F-ara-ATP was eliminated from the treated enzyme; however, the addition of pyrophosphate, followed by gel filtration chromatography, restored enzyme activity, indicating that F-ara-ATP bound to the enzyme and altered the AMP-binding site. Secondly, the activity of DNA ligase I was inhibited when F-ara-ATP was incorporated into the 3' terminus of the DNA substrate. The dual mode of inhibition of DNA ligase I by F-ara-ATP indicates that its effect on DNA ligation may be important in the inhibition of DNA synthesis and the cytotoxicity of F-ara-A.

Adenosine Monophosphate↗
PubMed1992

Participation of pertussis toxin-sensitive GTP-binding regulatory proteins in the suppression of baroreceptor reflex by neurotensin in the rat.

We evaluated the molecular mechanism that may underlie the suppressive effect of neurotensin (NT) on the baroreceptor reflex (BRR), using Sprague-Dawley rats that were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of NT (15 nmol) significantly inhibited the BRR response. Such an inhibition was appreciably antagonized by pretreating animals with i.c.v. injection of pertussis toxin (10 or 20 pmol), N-ethylmaleimide (1 or 2 nmol), forskolin (30 or 60 nmol) or phorbol 12-myristate 13-acetate (2 or 4 nmol), but not by cholera toxin (15 or 30 pmol). More specifically, pretreatments with bilateral microinjection into the nucleus tractus solitarius (NTS) of pertussis toxin (80 or 160 fmol), N-ethylmaleimide (80 pmol), forskolin (480 pmol) or phorbol 12-myristate 13-acetate (16 or 32 pmol) also blunted the NT-induced suppression of BRR, although cholera toxin (120 or 240 fmol), or 1,9-dideoxyforskolin (480 pmol) had no appreciable effect. These results suggest that a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein(s), which is not likely to be Gs, possibly Gi or Gp, may be involved in the transmembrane signaling process that underlies the suppression of BRR response by NT at the NTS.

Animals↗
PubMed1992

Reversal by pertussis toxin and N-ethylmaleimide of the facilitation of baroreceptor reflex response by somatostatin in the rat.

We evaluated the transmembrane signaling mechanism that may underlie the facilitatory action of somatostatin (SOM) on baroreceptor reflex (BRR), using adult, male, Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of SOM (2 nmol) promoted a significant elevation in BRR response, induced by phenylephrine (5 micrograms/kg, i.v.). This potentiatory action of the tetradecapeptide was significantly reversed after pretreating animals with bilateral microinjection of pertussis toxin (25 ng) or N-ethylmaleimide (2 nmol) into the nucleus tractus solitarius (NTS), the terminal site for baroreceptor afferents. These results suggest that a pertussis toxin-sensitive GTP-binding regulatory protein, possibly Gi, may be involved in the modulation of the BRR by SOM at the NTS.

Afferent Pathways↗
PubMed1992

Left ventricular opacification after peripheral venous injection of a modified albumin solution.

The usefulness of a modified albumin solution was assessed in 8 dogs after peripheral venous and inferior vena cava injections. The contrast agent is a mixed solution made of glucose, albumin and glycerin, with sonicated microbubble diameter of 5.0 +/- 2.3 microns. Multiple injections (8 ml each) of this contrast agent (total 80 injections) into peripheral vein and inferior cava were performed. The blood pressure from femoral artery was measured before, during and after injections. Two-dimensional echocardiograms were recorded in a modified long axis view on videotapes for play back analysis. The pulmonary transit time and left ventricular contrast persistent time was determined for each injection. The videodensity of the region of interest (ROI) at the center of right ventricle and left ventricle was measured. The background videodensity of both ventricles was evaluated. The videodensity over the ROI of both ventricles with peak contrast enhancement was measured in all frames for 3 consecutive cardiac cycles. The peak videodensity of right and left ventricle subtracting the background videodensity of each ventricles was further calculated respectively. The injections caused no change in blood pressure or heart rate. All injections produced right ventricular contrast echo. As much as 85% of peripheral venous and 82.5% of inferior vena cava injections resulted in left ventricular contrast which was 0.68 and 0.65 as bright as that produced in the right ventricle. Pulmonary transit time and left ventricle contrast persistent time of peripheral venous injection was 4.05 +/- 0.53 and 13.67 +/- 4.28 seconds respectively. No difference of these data (3.93 +/- 0.47 and 11.65 +/- 4.66 seconds) from those produced by inferior vena cava injections were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1992

Intracavernous pressure as an experimental index in a rat model for the evaluation of penile erection.

This report communicates our attempt to design a small animal model for the evaluation of penile erection, based on the pharmacological responses of cavernous tissues in the rat that resemble those of human subjects. Male adult Sprague-Dawley rats anesthetized with pentobarbital sodium were used in conjunction with papaverine and prostaglandin E1, two vasoactive drugs most commonly used in clinical management of impotence. Intracavernous administration of papaverine (0.05, 0.1, 0.02, 0.4 or 0.8 mg.) induced a progressive increase in intracavernous pressure that peaked at 0.4 mg. This effect was associated with visible penile erection that became conspicuous when accompanied by additional bursts of transient intracavernous pressure fluctuations. The duration of papaverine-induced increase in intracavernous pressure was significantly shortened by clonidine (15 micrograms, intracavernous). Injection of prostaglandin E1 (1, 2 or 4 micrograms) into the corpus cavernosum also elicited an elevation in intracavernous pressure, but the responses exhibited acute tachyphylaxis. By manifesting a response to papaverine and prostaglandin E1 that is similar to that in human, we conclude that the intracavernous pressure in the rat may represent a suitable index for the evaluation of penile erection in small laboratory animals.

Alprostadil↗
PubMed1992

Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions.

The incorporation of 6-thioguanine (S6G) in place of guanine proceeds readily in DNA synthesis reactions catalyzed by mammalian and bacterial polymerases. This report summarizes the consequences of such incorporation studied to date. S6G was incorporated into one strand of a defined M13mp18 phage sequence in a (+)reaction catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. After denaturation of the newly synthesized strand (containing S6G) and annealing with a reverse (-) 32P-labeled primer, polymerization catalyzed by the Klenow enzyme as well as by human DNA polymerases alpha, gamma, and delta was slowed considerably, compared with that across the corresponding guanine-containing template. To evaluate S6G-containing DNA as a substrate for DNA ligases, two oligodeoxynucleotides (19- and 20-mers) antisense to a 40-mer were synthesized so that the 40-mer coded for guanine at the 3' terminus of the 19-mer. After annealing of the synthetic oligonucleotides to form a duplex DNA containing a one-nucleotide gap (opposite cytosine in the 40-mer), the 19-mer was extended with 2'-deoxythioguanosine 5'-triphosphate using DNA polymerase, forming a nicked duplex DNA. The abilities of T4 DNA ligase and HeLa and calf thymus DNA ligase I to join the 5'-phosphate with the 3'-S6G-OH were severely inhibited, compared with the 3'-guanine-extended control. This finding suggests that incorporation of S6G at the 3' terminus of Okazaki fragments would inhibit lagging strand DNA synthesis. In other experiments, cleavage of S6G-containing DNA by some but not all restriction endonucleases progressed poorly, compared with the control guanine-containing DNA, independently of the location of S6G at recognition or cleavage sites, as previously observed by Iwaniec et al. [Mol. Pharmacol. 39:299-306 (1991)] with a different spectrum of enzymes. These findings indicate altered DNA-protein interactions due to S6G incorporation. The poor template function of S6G-containing DNA is consistent with the known delayed cytotoxicity and DNA damage previously reported to occur in S6G-treated cells.

Autoradiography↗
PubMed1991

Reduction in postsynaptic alpha 2-adrenoceptor activity by endogenous angiotensin III in the nucleus reticularis gigantocellularis of the rat.

We investigated in adult, male Sprague-Dawley rats anesthetized with pentobarbital sodium the synaptic location of the interaction between endogenous angiotensin III (AIII) and the alpha 2-adrenoceptors in the medulla oblongata that are involved in cardiovascular regulation. The circulatory suppressant efficacy of a centrally acting alpha 2-adrenoceptor agonist, guanabenz, was used as the experimental index. Direct bilateral microinjection of AIII (40 pmol) into the nucleus reticularis gigantocellularis (NRGC), a medullary site believed to be intimately related to the cardiovascular inhibitory actions of guanabenz, attenuated, whereas the selective AIII receptor antagonist, Ile7-AIII (20 nmol), potentiated, the circulatory suppressant effects of guanabenz (100 micrograms/kg, i.v.). These two respective actions were essentially unaffected by immunocytochemically verified depletion of noradrenergic nerve terminals in the NRGC, elicited by a selective noradrenergic neurotoxin, DSP4. These data suggest that endogenous AIII may exert a tonic inhibitory action on the alpha 2-adrenoceptors located postsynaptically on neurons in the NRGC that are involved in central cardiovascular regulation.

Adrenergic alpha-Antagonists↗
PubMed1991

Facilitation of baroreceptor reflex response by endogenous somatostatin in the rat.

We evaluated the potential participation of endogenous brain somatostatin-14 (SOM) in central cardiovascular regulation, using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of SOM (2 or 4 nmol) promoted a significant elevation in baroreceptor reflex (BRR) response, induced by phenylephrine (5 micrograms kg, i.v.). Blocking the endogenous SOM activity with its specific receptor antagonist, cyclo-[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)] (2 or 4 nmol, i.c.v.) or antiserum against SOM (1:20, i.c.v.), on the other hand, appreciably attenuated the same response. These modulatory effects on the BRR response were essentially duplicated upon bilateral microinjections of SOM (320 pmol), SOM antagonist (320 pmol) or anti-SOM (1:20) into the caudal portion of the nucleus of tractus solitarius (NTS), the terminal site for baroreceptor afferents. These results suggest that neurons that contain SOM may participate in cardiovascular control by tonically facilitating the BRR, possibly by exerting an influence on the neurons at the NTS.

Animals↗
PubMed1991

Tissue- and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1.

Protein 4.1, a multifunctional structural protein originally described as an 80-kDa component of the erythroid membrane skeleton, exhibits tissue- and development-specific heterogeneity in molecular weight, subcellular localization, and primary amino acid sequence. Earlier reports suggested that some of this impressive heterogeneity is generated by alternative RNA splicing (Conboy, J. G., Chan, J., Mohandas, N., and Kan, Y. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9062-9065; Tang, T. K., Leto, T., Marchesi, V. T., and Benz, E. J. (1990) J. Cell Biol. 110, 617-624). We have now completed a systematic analysis of 4.1 mRNA isoforms expressed in erythroid cells, and have generated an "alternative splicing map" which summarizes diagrammatically a multitude of polypeptide isoforms potentially generated by combinatorial splicing of nine alternative exons. Complex 5' splicing events yield mRNA isoforms that may initiate translation at different sites and thus generate elongated or truncated NH2 termini; elongated approximately 135-kDa and prototypical approximately 80-kDa species were detected in both erythrocytes and T-lymphocytes, but in very different ratios. Among the functional domains of 4.1 responsible for interaction with other membrane skeletal elements, four variants of the 10-kDa spectrin-actin-binding region and four variants of the putative 30-kDa glycophorin-binding region are predicted. Developmentally controlled alternative RNA splicing in the spectrin-actin-binding region may help regulate remodeling of membrane architecture and mechanical properties that occur during erythropoiesis.

Amino Acid Sequence↗
PubMed1991

Coordinate activation of multidrug-resistance (P-glycoprotein) genes mdr2 and mdr3 during mouse liver regeneration.

We previously reported that only one of the three mouse multidrug-resistance (mdr) genes, mdr3, is activated in hepatocellular carcinomas (HCCs). The present study examined the expression of mdr family members during mouse liver regeneration after partial hepatectomy to determine whether the regeneration that occurs during hepatic tumorigenesis is responsible for mdr3 elevation in HCC. We demonstrated that in both C3H/HeN and B6C3/F1 mice strains, the levels of both mdr2 and mdr3 mRNAs coordinately increased five- to sevenfold 24 h after partial hepatectomy, whereas the levels of mdr1 mRNA were not statistically different from those in the controls. Forty-eight hours after partial hepatectomy, mdr mRNA levels decreased and in most cases returned to normal levels after 72 h. These results indicate that mdr3 induction during hepatocarcinogenesis is not due to liver regeneration alone.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Participation of noradrenergic neurotransmission in angiotensin III-induced dipsogenic behavior in the rat.

Conscious, adult, male Sprague-Dawley rats, instrumented with in-dwelling cannula for drug application into the lateral cerebral ventricle, were used to evaluate the participation of noradrenergic neurotransmission in angiotensin III (AIII)-induced dipsogenic behavior. Intracerebroventricular (i.c.v.) administration of AIII (20, 40 or 80 pmol) elicited a robust and dose-related drinking response. Chemical lesion produced by i.c.v. injection of the catecholaminergic neurotoxin, 6-hydroxydopamine (25 micrograms x 3), or the selective noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (250 micrograms), promoted significant antagonization of the dipsogenic behavior produced by AIII (40 or 80 pmol, i.c.v.). Under equimolar doses (3.25 or 6.50 nmol), the specific alpha 1-adrenoceptor blocker, prazosin, antagonized; the specific alpha 2-adrenoceptor antagonist, yohimbine, enhanced; and the nonselective alpha-adrenoceptor blocker, phentolamine, elicited minimal action, on AIII (40 pmol)-induced drinking response. These results suggest that central noradrenergic neurotransmission may participate actively in AIII-induced dipsogenesis, in a process that may involve both alpha 1- and alpha 2-adrenoceptors.

Adrenergic alpha-Antagonists↗
PubMed1991

Locus coeruleus control of spinal motor output.

Using electrophysiological techniques, we investigated the functional properties of the coeruleospinal system for regulating the somatomotor outflow at lumbar cord levels. Many of the fast-conducting, antidromically activated coeruleospinal units were shown to exhibit the alpha 2-receptor response common to noradrenergic locus coeruleus (LC) neurons. Electrically activating the coeruleospinal system potentiated the lumbar monosynaptic reflex and depolarized hindlimb flexor and extensor motoneurons via an alpha 1-receptor mechanism. The latter synaptically induced membrane depolarization was mimicked by norepinephrine applied iontophoretically to motoneurons. That LC inhibited Renshaw cell activity and induced a positive dorsal root potential at the lumbar cord also reinforced LC's action on motor excitation. We conclude that LC augments the somatomotor output, at least in part, via an alpha 1-adrenoceptor-mediated excitation of ventral horn motoneurons. Such process is being strengthened by LC's suppression of the recurrent inhibition pathway as well as by its presynaptic facilitation of afferent impulse transmission at the spinal cord level.

Action Potentials↗
PubMed1991

Effects of adrenalectomy and nephrectomy on adrenal regeneration hypertension in the rat.

The involvement of the regenerating adrenal gland and kidney, and the contribution of deoxycorticosterone (DOC) and prostaglandin E2 (PGE2), in the development of adrenal regeneration hypertension (ARH) was evaluated in young female Sprague-Dawley rats. Based on tail-cuff plethysmographic measurement, animals subjected to nephrectomy and adrenalectomy on the right side and adrenal enucleation (removal of the adrenal cortex) on the left side developed significant (P less than 0.05, n = 12) hypertension within 6 weeks following operation. Subsequent left nephrectomy in these ARH rats produced a further elevation, whereas a secondary adrenalectomy resulted in an acute and discernible reduction in blood pressure within 24-36 hours. It is interesting to note that the progressive increase in blood pressure following left nephrectomy was significantly reversed by PGE2 (10 or 20 micrograms/kg, i.p.). At the same time, the reduction in blood pressure after secondary adrenalectomy was significantly retarded by deoxycorticosterone trimethylacetate (2 mg/kg, i.p.). These data demonstrated that both the kidney and the regenerating adrenal cortex are involved in the pathogenesis of ARH. Furthermore, it is probable that the secretion of DOC by the regenerating adrenal cortex is responsible for the elevation in blood pressure, in a process that is balanced by PGE2, possibly secreted by the kidney.

Adrenal Glands↗
PubMed1990

Fingerprinting of near-homogeneous DNA ligase I and II from human cells. Similarity of their AMP-binding domains.

DNA ligases play obligatory roles during replication, repair, and recombination. Multiple forms of DNA ligase have been reported in mammalian cells including DNA ligase I, the high molecular mass species which functions during replication, and DNA ligase II, the low molecular mass species which is associated with repair. In addition, alterations in DNA ligase activities have been reported in acute lymphocytic leukemia cells, Bloom's syndrome cells, and cells undergoing differentiation and development. To better distinguish the biochemical and molecular properties of the various DNA ligases from human cells, we have developed a method of purifying multiple species of DNA ligase from HeLa cells by chromatography through DEAE-Bio-Gel, CM-Bio-Gel, hydroxylapatite, Sephacryl S-300, Mono P, and DNA-cellulose. DNA-cellulose chromatography of the partially purified enzymes resolved multiple species of DNA ligase after labeling the enzyme with [alpha-32P]ATP to form the ligase-[32P]AMP adduct. The early eluting enzyme activity (0.25 M NaCl) contained a major 67-kDa-labeled protein, while the late eluting activity (0.48 M NaCl) contained two major labeled proteins of 90 and 78 kDa. Neutralization experiments with antiligase I antibodies indicated that the early and late eluting activity peaks were DNA ligase II and I, respectively. The three major ligase-[32P]AMP polypeptides (90, 78, and 67 kDa) were subsequently purified to near homogeneity by elution from preparative sodium dodecyl sulfate-polyacrylamide gels. All three polypeptides retained DNA ligase activities after gel elution and renaturation. To further reveal the relationship between these enzymes, partial digestion by V8-protease was performed. All three purified polypeptides gave rise to a common 22-kDa-labeled fragment for their AMP-binding domains, indicating that the catalytic sites of ligase I and II are quite similar, if not identical. Similar findings were obtained from the two-dimensional gel electrophoresis of their AMP-binding domains in the trypsin-digested protein fragments. The results also suggested that these isozymes have been derived from the same primordial DNA sequence or from the same precursor protein. The purification scheme and the data obtained will be instrumental for the further elucidation of the biological roles of various DNA ligases from human cells.

Adenosine Monophosphate↗
PubMed1990

Tonic enhancement of the sensitivity of baroreceptor reflex response by endogenous substance P in the rat.

Modulation of baroreceptor reflex (BRR) by endogenous substance P (SP) in the brain was investigated in rats anesthetized with pentobarbital sodium. Intracerebroventricular administration of the undecapeptide (15 or 30 nmol) and its antagonist, (D-Pro2, D-Trp7,9)-SP (30 or 60 nmol) or SP antiserum (1:20), respectively, promoted a significant increase and decrease in the sensitivity of BRR response. Prolonging the endogenous activity of SP with the aminopeptidase blocker, bestatin (200 nmol) or with the endopeptidase-24.11 inhibitor, phosphoramidon (200 nmol) significantly augmented the same reflex. Combining the undecapeptide with either peptidase blocker, moreover, promoted additional potentiation of the BRR response. On the other hand, simultaneous administration of bestatin and (D-Pro2, D-Trp7,9)-SP produced a reduction of the augmented effect of bestatin on the sensitivity of BRR response. Bilateral microinjection of SP (600 pmol) or an antiserum against SP (1:20) into the nucleus tractus solitarius (NTS) elicited respectively an enhancement of and reduction in the BRR response. These data suggest that neurons that contain SP may participate in central cardiovascular control by tonically enhancing the sensitivity of the BRR response, possibly via an action on the NTS.

Animals↗
PubMed1990

Tonic suppression of baroreceptor reflex by endogenous neurotensin in the rat.

We evaluated the modulatory role of endogenous neurotensin (NT) in baroreceptor reflex (BRR) response in Sprague-Dawley rats anesthetized with pentobarbital sodium. Intracerebroventricular (i.c.v.) administration of NT (15 or 30 nmol) significantly reduced the sensitivity of the BRR response. Blocking the endogenous activity of the tridecapeptide with its specific antagonist, (D-Trp11)-NT (4 or 8 nmol) or antiserum against NT (1:20); or inhibiting the aminopeptidases with bestatin (200 nmol), on the other hand, promoted a potentiation of BRR response. When administered together with bestatin (200 nmol), the suppressive effect of NT (15 nmol) on the BRR response was further enhanced, as was the augmentative action of (D-Trp11)-NT (4 nmol). Upon microinjection into the bilateral nucleus tractus solitarius (NTS), NT (600 pmol) and (D-Trp11)-NT (150 pmol) respectively elicited a reduction and enhancement of the BRR response. These results suggest that neurons that contain NT may participate in central cardiovascular regulation by tonically suppressing the BRR, possibly via an action on the NTS where baroreceptor afferents terminate.

Animals↗