Biomedical subjects

B H Lee

Publications and source records attributed to B H Lee.

At least 19 recordsLinked to original sources

PubMed1999

Manganese-containing superoxide dismutase and its gene from Candida albicans.

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.

Amino Acid Sequence↗

PubMed1999

The nuclei innervating digastric muscle do not project to the hypoglossal nucleus in the rat.

We employed the retrograde neural tracing method using the pseudorabies virus bartha strain (PRV-Ba) to investigate the projection from accessory trigeminal nucleus (Acc5) or accessary facial nucleus (Acc7) to hypoglossal nucleus (HP). After injection of PRV-Ba into the bellies of the digastric muscle, the PRV-Ba containing neurones were observed in Acc5 of the cranial belly injected rats and Acc7 of caudal belly injected rats ipsilaterally, but not in HP. These results suggest that Acc5 and Acc7 may not project to HP and that movements of the digastric muscle are not related to cooperation with tongue movements during mastication.

Animals↗

PubMed1999

Characterization of angiotensin II antagonism displayed by SK-1080, a novel nonpeptide AT1-receptor antagonist.

The pharmacologic profile of SK-1080, a nonpeptide AT1-selective angiotensin-receptor antagonist, was investigated by receptor-binding studies, functional in vitro assays with rabbit and rat aorta, and in vivo experiments in pithed rats. SK-1080 inhibited the specific binding of [125I]-[Sar1, Ile8]-angiotensin II to human recombinant AT1 receptor with a 12-fold greater potency than losartan [median inhibitory concentration (IC50): 1.01 and 12.3 nM, respectively], but it did not inhibit the binding of [125I]-CGP 42112A to human recombinant AT2 receptor (IC50: >10 microM for both). The Hill coefficient for the competition curve of SK-1080 against AT1 receptor was not significantly different from unity (0.96). Scatchard analysis showed that SK-1080 interacted with human recombinant AT1 receptor in a competitive manner, as with losartan. In functional studies with rat and rabbit aorta, SK-1080 competitively inhibited the contractile response to angiotensin II (pKB values: 9.97 and 9.51, respectively) with 15-25% decrease in the maximal contractile responses, unlike losartan, which showed competitive antagonism without any change in the maximal contractile responses to angiotensin II (pA2 values, 8.02 and 7.59, respectively). In pithed rats, SK-1080 (i.v.) induced a nonparallel right shift in the dose-pressor response curve to angiotensin II (ID50, 0.07 mg/kg) with a dose-dependent reduction in the maximal responses; this antagonistic effect was approximately 25 times more potent than losartan (ID50, 1.74 mg/kg), which showed competitive antagonism. SK-1080 did not alter the responses induced by other agonists such as norepinephrine, KCI, and vasopressin in isolated rabbit aorta and pithed rats. These results suggest that SK-1080 is a highly potent AT1-selective angiotensin II-receptor antagonist with a mode of insurmountable antagonism.

Angiotensin II↗

PubMed1999

In vivo pharmacologic profile of SK-1080, an orally active nonpeptide AT1-receptor antagonist.

The pharmacologic profile of SK-1080, a newly synthesized AT1-receptor antagonist, was evaluated in conscious normotensive rats, conscious renally (RHRs) and spontaneously (SHRs) hypertensive rats, and conscious furosemide-treated beagle dogs. In angiotensin II-challenged normotensive rats, orally administered SK-1080 had no agonistic effect and dose-dependently inhibited the pressor response to angiotensin II with a slightly weaker potency (ID50: 1.12 and 0.47 mg/kg, respectively), but with a more rapid onset of action than losartan (time to Emax, 30 min and 6 h, respectively). In RHRs, orally given SK-1080 produced a dose-dependent and long-lasting (>24 h) antihypertensive effect with a potency similar to that of losartan (ED20, 5.06 and 3.36 mg/kg, respectively). Intravenously administered SK-1080 exerted a very highly potent antihypertensive effect (ED20, 0.06 mg/kg), thus indicating a poor oral bioavailability in rats. On repeated dosing for 21 days in SHRs, SK-1080 significantly reduced blood pressure without inducing tachycardia and tolerance throughout the dosing period. On repeated dosing, the antihypertensive effect gradually increased from days 1 to 7 (Emax on day 7, 15.0 and 19.7% at 10 and 30 mg/kg, respectively) and remained at a significant level on days 14 and 21. In furosemide-treated dogs, orally given SK-1080 produced a dose-dependent and long-lasting (>8 h) antihypertensive effect with a rapid onset of action (time to Emax, 1-1.5 h) and 10-fold greater potency than losartan (ED20, 0.72 and 8.13 mg/kg, respectively). In furosemide-treated dogs, SK-1080 showed a good oral bioavailability, unlike that in RHRs. These results suggest that SK-1080 is a potent, orally active AT1-receptor antagonist useful for the treatment of hypertension.

Administration, Oral↗

PubMed1999

Proposing sequences for peptides derived from whey fermentation with potential bioactive sites.

In fed-batch fermentation by Kluyveromyces marxianus var. marxianus, whey-soluble proteins were converted into oligopeptides. To assess whether bioactive peptides could be produced during whey fermentation, K. marxianus was cultured in batch in deproteinized media containing 5 or 15% (wt/vol) dehydrated whey for 20 h and then was in fed-batch mode for 50 h. After harvesting the biomass (25,000 x g, 15 min), at 6-h intervals, the wort was analyzed to determine protein consumption and oligopeptide production by HPLC. The proteins in the wort showed an oscillatory degradation with a constant increase in the production of oligopeptides. Four major peaks were collected and were analyzed by API mass spectroscopy. Sequences of fermented peptides were compared with sequences of known bioactive peptides. On the basis of their molecular weights, two amino acid sequences were proposed. The presence of sites containing the peptide sequence of beta-lactorphin (YLLF) suggests that these oligopeptides may have antihypertensive properties.

Amino Acid Sequence↗

PubMed1999

Preventive effects of a mixed disulphide from dithiocarbamate and N-acetylcysteine on the genotoxicity of N-nitrosodiethylamine.

A mixed disulphide model compound, S-(N,N-diethyldithiocarbamoyl)-N-acetylcysteine (AC-DDTC), prepared from diethyldithiocarbamate and N-acetylcysteine, was investigated for protective effects against the genotoxicity of the environmental carcinogen N-nitrosodiethylamine (NDEA). AC-DDTC was found to be a potent inhibitor of genotoxicity induced by NDEA. The mutagenicity of NDEA to Salmonella typhimurium was inhibited by 70% at 3.2 micromol AC-DDTC per plate and the effect was dose-dependent. In the chromosome aberration assay, NDEA elicited a significant increase in the number of aberrant cells. Pretreatment with AC-DDTC suppressed the chromosome-damaging effect of NDEA. The micronucleus-inducing capacity of NDEA was reduced by 32% by treatment with AC-DDTC at 1.5 mmol kg(-1). These results suggest that AC-DDTC might have a role to play in reducing the risk of cancer induced by NDEA.

Acetylcysteine↗

PubMed1998

Regulation of fibronectin gene expression by cyclic AMP and phorbol myristate acetate in HT-1080 human fibrosarcoma cells.

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.

Blotting, Northern↗

PubMed1998

D-arabinose dehydrogenase and its gene from Saccharomyces cerevisiae.

D-Arabinose dehydrogenase was purified 843-fold from the cytosolic fraction of Saccharomyces cerevisiae with a recovery of 9%. The purified enzyme gave two bands with a molecular mass of 40 and 39 kDa on SDS-PAGE. The native enzyme had a molecular mass of 74 kDa as estimated by Sephacryl S-200 chromatography. Therefore, this enzyme was considered to be a heterodimer. The purified enzyme exhibited maximum activity at pH 10.0 and around 30 degrees C. The enzyme catalysed the oxidation of D-arabinose, L-xylose, L-fucose and L-galactose in the presence of NADP+. The apparent Km values at pH 10.0 with 50 microM NADP+ for D-arabinose, L-xylose, L-fucose, and L-galactose were 161, 24, 98 and 180 mM, respectively. The pH profile of Vmax and kcat/Km showed one ionisable groups around pH 8.3. D-Erythroascorbic acid was formed in vitro from D-arabinose by D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase. The N-terminal amino acid sequence of the heavy subunit was Ser-Thr-Glu-Asn-Ile-Val-Glu-Asn-Met-Leu-His-Pro-Lys-Thr-. The N-terminus of the light subunit was blocked. The obtained peptide sequence was identical to the translational product of an unknown open reading frame, YBR149W, in chromosome II of S. cerevisiae. When compared with the translational product of this open reading frame, the peptide sequence was identical to the amino acid sequences of residues 7 to 20. The first six amino acids of this open reading frame were lost in protein sequence, which may be modified post-translationally. The heavy subunit was composed of 344 amino acid residues and its deduced amino acid sequence contained the motifs I, II, and III of aldo-keto reductase and also leucine zipper motif. This enzyme is the first heterodimeric protein of aldo-keto reductase family. In the deletion mutant of this gene, D-arabinose dehydrogenase activity and D-erythroascorbic acid were not detected.

Alcohol Oxidoreductases↗

PubMed1998

Anthelmintic beta-hydroxyketoamides (BKAs).

We have prepared several anthelmintic coumarins based on the beta-hydroxyketoamide (BKA) template and have shown that this template remains valid over a wide range of changes to the coumarin moiety allowing for the inclusion of carbocyclic, bicyclic, and heterocyclic rings.

Animals↗

PubMed1998

C24 and C25 substituted marcfortine A derivatives.

The dioxepinoindole ring found in marcfortine A (1) is unique among natural products. In order to determine the importance of the substitution pattern of the C24-C25 olefin, we synthesized a variety of analogs at these positions. With the exception of compound 5, none of these compounds exhibited any anthelmintic activity.

Animals↗

PubMed1998

In vitro chromosome aberration assay using human bronchial epithelial cells.

The determination of chromosome aberrations (CA) in Chinese hamster lung fibroblast (CHL) cells was compared with that in normal human bronchial epithelial (BEAS-2B) cells, which upon adenovirus infection were reported to possess carcinogen metabolizing capacities similar to polycyclic aromatic hydrocarbons. CHL and BEAS-2B cells were treated with increasing concentrations of benzo[a]pyrene (BaP) or N-nitrosodiethylamine (NDEA). In BEAS-2B cells, BaP, at a concentration of 50 microg/ml, produced a significant increase in the CA frequency, while NDEA did not markedly alter the number of aberrations in the absence of S9 mixture. The CHL cells exposed to BaP and NDEA in the presence of S9 mixture responded as anticipated with a 30% and 14% frequency of CA observed in the BaP (50 microg/ml) and NDEA (1000 microg/ml) treated cells, respectively. The results of this study show that the CA assay using human cell line with intrinsic metabolic activation system, such as BEAS-2B cells, may be a useful model for predicting human clastogens and carcinogens.

Animals↗

PubMed1998

Inhibition of electric eel acetylcholinesterase by porphin compounds.

Synthetic porphin compounds have been found to be reversible inhibitors of acetylcholinesterase from electric eel with Ki values of microM range. It seems that the number and position of fluorine on the phenyl ring and metal of an inhibitor play an important role for binding of an inhibitor to the enzyme active site.

Acetylcholinesterase↗

PubMed1998

Oxygen depletion induced dormancy in Mycobacterium smegmatis.

We report here that the physiological behaviour of the fast growing saprophytic Mycobacterium smegmatis under in vitro oxygen-depletion and reactivation conditions is strikingly similar to the characteristics shown by the slow growing pathogenic M. tuberculosis. M. smegmatis died rapidly when shifted abruptly from aerobic to anaerobic conditions. In contrast to the lethal shock of abrupt oxygen depletion, the slow depletion through a self generated oxygen gradient permitted an adaptation to a persistent state which showed increased resistance against the bactericidal effects of anaerobiosis. The anaerobic persistent culture did not synthesise DNA and showed synchronised division upon reactivation in oxygen rich medium, indicating that the persistent bacilli are uniformly arrested at a defined stage of the cell cycle. Upon reactivation the persistent culture started synthesising DNA only after the first cell division, suggesting that the persistent cells contain two chromosomes. Furthermore, the persistent culture developed sensitivity to metronidazole and resistance against ofloxacin. These results suggest that M. smegmatis might be useful as a fast growing non-pathogenic model for comparative molecular analyses of mycobacterial dormancy.

Aerobiosis↗

PubMed1998

A simple program to calculate codon bias index.

A computer program (PCBI) was developed to quickly calculate codon bias index (CBI). PCBI can analyze a gene containing introns. The 22 preferred codons defined from Saccharomyces cerevisiae were used in PCBI as the standard to measure the CBI values. However, users can modify the preferred codons to suit each organism. The data PCBI provides include DNA sequence of open reading frame without introns, amino acid sequence of gene product, a table of amino acid composition, a table of codon usage and (G + C) content, parameters for calculating CBI, and the value of CBI. PCBI runs on a DOS or Windows environment, but results can be saved in ASCII text format.

Amino Acids↗

PubMed1998

Comparison of sympathetic sprouting in sensory ganglia in three animal models of neuropathic pain.

Sympathetic postganglionic fibers sprout in the dorsal root ganglion (DRG) after peripheral nerve injury. Therefore, one possible contributing factor of sympathetic dependency of neuropathic pain is the extent of sympathetic sprouting in the DRG after peripheral nerve injury. The present study compared the extent of sympathetic sprouting in the DRG as well as in the injured peripheral nerve in three rat neuropathic pain models: (1) the chronic constriction injury model (CCI); (2) the partial sciatic nerve ligation injury model (PSI); and (3) the segmental spinal nerve ligation injury model (SSI). All three methods of peripheral nerve injury produced behavioral signs of ongoing and evoked pain with some differences in the magnitude of each pain component. The density of sympathetic fibers in the DRG was significantly higher at all examined postoperative times than controls in the SSI model, while it was somewhat higher than controls only at the last examined postoperative time (20 weeks) in the CCI and PSI models. Therefore, data suggest that, although sympathetic changes in the DRG may contribute to neuropathic pain syndromes in the SSI model, other mechanisms seem to be more important in the CCI and PSI models at early times following peripheral nerve injury.

Animals↗

PubMed1998

Expression of gonadotropin-releasing hormone (GnRH) and GnRH receptor mRNA in prostate cancer cells and effect of GnRH on the proliferation of prostate cancer cells.

The purpose of this study was to determine the production of gonadotropin-releasing hormone (GnRH), the co-occurrence of GnRH receptors in prostate cancer cells, and the effect of GnRH on prostate cancer cell proliferation. Four human prostate cancer cell lines were studied. LNCaP is an androgen sensitive prostate cancer cell line, DU-145 and PC-3 are androgen resistant, and TSU-Pr1 is uncharacterized. The expression of GnRH and GnRH receptor mRNAs were assessed by in situ hybridization and the effect of exogenous GnRH on proliferation of prostate cancer cells was measured by thymidine incorporation assay. GnRH mRNA expression, determined by in situ hybridization, was found in 83.48% of the LNCaP, 89.7% of the TSU-Pr1, 86.2% of the PC-3 and 95.3% of the DU-145. Signals of GnRH receptor mRNA were detected in more than 95% of the cells of all four cell lines. The proliferation of the prostate cancer cells grown in media supplemented with peptide hormone lacking charcoal-stripped serum was significantly (P < 0.05) suppressed. No significant effect of GnRH on the proliferation of all four prostate cancer cells was observed. In summary, prostate cancer cells produced GnRH and its receptors, and exogenous GnRH treatment did not affect the prostate cancer cell proliferation. The existence of GnRH and GnRH receptor mRNA in the same cell suggests that the role of GnRH produced by prostate cancer cells would be autocrine.

Cell Division↗

PubMed1998

Upregulation of a histone-like protein in dormant Mycobacterium smegmatis.

The aerobic saprophyte Mycobacterium smegmatis, like its pathogenic counterpart M. tuberculosis, has the ability to adapt to anaerobiosis by shifting down to a dormant state. Here, we report the identification and molecular genetic characterisation of the first dormancy-induced protein in M. smegmatis. Comparative SDS-polyacrylamide gel electrophoresis of protein extracts of aerobically growing and dormant anaerobic M. smegmatis cultures revealed the upregulation of a 27-kDa protein in the dormant state. Peptide sequencing showed that the induced protein is a homologue of the histone-like protein H1p, predicted by the M. tuberculosis genome project. The corresponding hlp gene was cloned from M. smegmatis and sequenced. Disruption of the hlp gene eliminated the histone-like protein but did not affect the viability of the dormant culture.

Amino Acid Sequence↗

PubMed1998

Postembryonic development of leucokinin-like immunoreactive neurons in the moth Spodoptera litura.

Antiserum to leucokinin I, a neuropeptide originally isolated from the cockroach Leucophaea maderae, was used for immunocytochemical labeling of neurons in the brain and ventral ganglia of the moth Spodoptera litura during postembryonic development. In the ventral ganglia, leucokinin-like immunoreactivity begins to occur in the abdominal ganglion A3 to A7 of first instar larva. One to two weakly labeled pairs of bilateral LK-LI cell bodies are located in the subesophageal ganglion of fourth to sixth instar larvae and in the abdominal ganglia A1 to A7 of second to sixth instar larvae. The abdominal ganglion A1 of fourth to sixth instar larvae and A8 of sixth instar larva each contain one weakly labeled pair of median LK-LI cell bodies. Two strongly labeled pairs of bilateral LK-LI neurons are found in A3 to A7 of third to sixth instar larvae. Abdominal ganglia A1 to A8 of prepupa, pupa and adult contain one to three weakly labeled pairs of bilateral LK-LI neurons. Two strongly labeled pairs of bilateral LK-LI neurons in each of the abdominal ganglia of larva, prepupa, pupa and adult send axons to the neuropil, and then each axon bifurcates into two axonal branches. Theses axonal branches from two bundles. From each of the two pairs of neurons an axon exits through the posterior ventral nerve (N2) which runs to the transverse nerve of the next posterior segment. In larval brains, 2-16 pairs of bilateral LK-LI cell bodies can be found together with LK-LI processes in the central neuropil. The larval brains show large changes in the number of LK-LI neurons throughout postembryonic development. The number of LK-LI cell bodies are reduced in number from sixth instar larval brain. Therefore, prepupal, pupal and adult brains contain a smaller number of LK-LI cell bodies. Two pairs of LK-LI median neurosecretory cells located immediately beside the pars intercerebralis in larval brains increase to three pairs in the 7-day-old pupal brain. In the adult, however, LK-LI median neurosecretory cells decrease to one pair.

Animals↗