Biomedical subjects

J L Ko

Publications and source records attributed to J L Ko.

36 records · Page 2Linked to original sources

PubMed1992

T-cell receptor beta-chain gene usage in the T-cell recognition of Mycobacterium leprae antigens in one tuberculoid leprosy patient.

The beta chain of the T-cell antigen receptor present on 20 T-cell clones isolated from a tuberculoid leprosy patient was studied by gene rearrangement and PCR analysis. These T-cell clones all responded to Mycobacterium leprae-encoded protein antigens, and 8 of them specifically recognized peptides of the mycobacterial 65-kDa heat shock polypeptide (65hsp). All T-cell clones studied were HLA-DR-restricted (DR2 or -3). In the DR3-restricted group, 7 of 10 used a beta-chain variable region V beta 5 gene family member, whereas in the DR2-restricted group, 2 of 10 T-cell clones used a V beta 5 gene segment and 5 used the V beta 18 gene segment. The deduced amino acid sequences of the beta chain from 8 T-cell clones have revealed that 3 of 4 DR3-restricted T-cell clones expressed the V beta 5.1 gene segment whereas the fourth DR3-restricted T-cell clone employed a V beta 5 family member not previously described. The V beta 5.1-positive T-cell clones all recognized the same 65hsp peptide from residues 2 to 12. The N-D-N segment (where D is diversity) of the junctional region of these T-cell clones was very similar, despite different beta-chain joining gene segments. Of the 4 DR2-restricted T-cell clones investigated, 3 used the V beta 18 gene segment and recognized the 65hsp peptide from residues 418 to 427. In conclusion, within this panel of M. leprae-reactive T-cell clones, the DR3-restricted T-cell clones mainly used a V beta 5 gene segment, whereas the DR2-restricted clones employed preferentially the V beta 18 gene segment.

Amino Acid Sequence↗

PubMed1992

Characterization of beta-125I-endorphin cross-linked proteins in NG108-15 cell membranes. A 25-kilodalton protein with properties of delta-opioid-binding site.

Cross-linking of beta-125I-endorphin to NG108-15 cell membranes labeled bands with molecular masses of 55, 35, and 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We applied several criteria to evaluate the relevance of these cross-linked bands to delta-opioid receptors, including selectivity, stereospecificity, affinity, G-protein coupling, down-regulation, and correlation with opioid receptor level in different well-characterized cell lines. Only the 25 kDa protein adequately fulfilled all these criteria. Thus, cross-linking to the 25-kDa band was selectively inhibited by ligands with delta-opioid affinity, but not by mu-opioid, kappa-opioid, or optically inactive opioid ligands or by non-opioid ligands. Based on inhibition of cross-linking, we calculated an affinity of [D-Ala2,D-Leu5]enkephalin binding to the 25-kDa and (Kd = 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased approximately 10-fold in the presence of Na+/guanyl-5'-yl imidodiphosphate. Chronic agonist treatment of NG108-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a manner parallel to down-regulation of opioid receptors. Finally, the amount of the 25-kDa band was roughly proportional to the level of opioid receptors present in N18TG2, NS20Y, ST7-3, and ST8-4 cells. The 25-kDa band was absent in PC12h, NIH3T3, and C6BU1 cells as well as in liver, all of which had no detectable opioid binding.

3T3 Cells↗

PubMed1992

Specific reduction of delta-opioid receptor binding in transfected NG108-15 cells.

We have recently identified and sequenced the cDNA for an opioid-binding protein with homologies to cell adhesion molecules (OBCAM) (Schofield, P. R., McFarlard, K. C., Hayflick, J. S., Wilcox, J. N., Cho, T. M., Roy, S., Lee, N. M., Loh, H. H., and Seeburg, P. H. (1989) EMBO J. 8, 489-495). Several lines of evidence using antibodies suggest that OBCAM may play a functional role in NG108-15 neuroblastoma x glioma cells, a useful model system that contains a homogeneous population of delta-opioid receptors. A logical extension of this research is to further test this hypothesis. As part of this study, NG108-15 cells were stably transfected with either sense or antisense sequences of a portion of pROM, the rat cDNA for OBCAM. [3H] Diprenorphine binding was greatly reduced in antisense-transfected cells relative to non-transfected cells. Binding to alpha 2-adrenergic, muscarinic, and insulin receptors was unaffected. These results further support the notion that OBCAM or its analogue is part (or a subunit) of an opioid receptor. Furthermore, our observation of an apparently specific reduction in opioid binding in these transfected cells suggests that they may provide a novel genetic approach for studying regulation of the opioid receptor in this defined cell line.

Amino Acid Sequence↗

PubMed1992

Expression of the adenovirus E1B 175R protein and its association with membranes of Escherichia coli.

The E1B 175-amino-acid (175R) protein of adenovirus 2 is required for cellular transformation of primary cells and establishing cell morphology in lytically infected cells. To investigate the biochemical function of this protein, we constructed a bacterial expression vector (pKHB1-T) to produce the 175R protein in sufficient amounts for purification and biochemical analysis. On the basis of DNA sequencing, gel electrophoresis, and immunoblot analysis, the pKHB1-T-encoded 175R protein appears to be identical to that expressed transiently in mammalian or adenovirus-transformed cells. The bacterially produced viral protein was also found to be quite stable and without any modifications. Partial purification of the pKHB1-T-encoded protein revealed that the majority of its associates with the inner membrane of the bacterial cell. This, together with the possibility of the 175R protein containing an N-terminal amphipathic alpha-helix as a potential translocation signal, suggests that there may be a common mechanism of protein transport operating in both eucaryotic and procaryotic systems.

Adenoviridae↗

PubMed1990

Analgesic, receptor binding, and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides.

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin1-5 sequences caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [3H]-dihydromorphine. The potency of receptor binding against [3H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100 mM), the effect of enhancing binding against [3H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid mu-receptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decreased in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.

Amino Acid Sequence↗

PubMed1989

Differential cytotoxicity of sodium arsenite in human fibroblasts and Chinese hamster ovary cells.

Human skin fibroblast (HF) cells were approximately 10-fold more sensitive to sodium arsenite toxicity than Chinese hamster ovary (CHO) cells using the clonogenic assay. G1-phase CHO cells showed a 2-fold increase in the susceptibility to the toxic effects of sodium arsenite as compared to asynchronous CHO cells. The concentrations of sodium arsenite required to kill 50% of the cell population were correlated with the intracellular glutathione levels in asynchronous, G1-phase CHO, and asynchronous HF cells. Moreover, verapamil potentiated the cytotoxicity of sodium arsenite in CHO cells but not in HF cells. These results indicated that a verapamil-sensitive outward channel may be involved in detoxification of arsenic in CHO cells. Treatment with sodium arsenite resulted in a marked cell-cycle disturbance in CHO, but not in HF cells. Thus, CHO cells may take time to recover from sodium arsenite insult before progressing through the cell cycle. A different response of sodium arsenite in heat-shock protein synthesis in these 2 cell types was also revealed.

Animals↗

PubMed1989

Two distinct immunogenic epitopes on the alpha chain of human T cell antigen receptor.

Monoclonal antibodies (MAb) to human T Cell Antigen Receptor (TCR) have been used to study the structure and function of TCR. Using purified alpha/beta heterodimeric protein, we have generated two MAb against human TCR alpha protein. The two MAb, alpha F1 and alpha F2, recognized amino acid residues 141-159 and 212-231 of the constant region of the alpha chain TCR. Although neither MAb reacted with viable T cells, both antibodies immunoprecipitated TCR alpha/beta heterodimer from HPB-ALL, Jurkat, PBL and a 32 kDa in vitro translation product of alpha chain cDNA. These antibodies have been shown to be useful in the immunohistochemical staining of human tissues. These two MAb, together with other anti-framwork MAb to TCR, should provide valuable reagents in the study of TCR and clinical classification of T cell lineage neoplasms.

Antibodies, Monoclonal↗

PubMed1988

Purification and characterization of a lethal protein with phospholipase A1 activity from the hornet (Vespa basalis) venom.

The hornet, Vespa basalis, is one of the most dangerous species of wasps found in Taiwan. The insect is aggressive and its venom is highly toxic. By gel filtration on a Fractogel TSK HW 50 column followed by cation-exchange chromatography on CM-Trisacryl M, a lethal protein was purified from the venom. It has a molecular mass of about 32 kDa and an i.v. LD50 value of 0.32 micrograms/g mouse. The toxin is capable of catalyzing the hydrolysis of emulsified phospholipids but not sphingomyelin. Analysis of the 1H-NMR spectra of the substrates and its hydrolytic products revealed that the toxin liberates fatty acid from the 1-position of sn-3-phosphoacylglycerols. This result indicates that the toxin possesses phospholipase A1 activity. The toxin exhibits an extremely potent hemolytic activity in washed red cells and diluted whole blood (HC50 = 0.09 micrograms/ml in mouse). The potency of direct hemolysis is about 100-times that of a basic phospholipase A2 from Naja nigricollis venom and about 1000-times that of a cardiotoxin from Naja naja atra venom. A positive correlation between the hemolytic activity and lethality of the toxin was found in three species of animals (mouse, rat and guinea pig). In the in vivo study, the toxin caused a marked increase in the plasma K+ concentration and a hyperkalemic change in the ECG of the treated rat. Hyperkalemia resulting from the hemolytic action of the toxin appears to be the main cause of death in the animal.

Amino Acid Sequence↗

PubMed1988

Beta-endorphin. Receptor binding and peripheral opioid activities of [Gln8]-, [Trp27]-, and [Tyr31]-analogs.

Three beta h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid mu-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln8]-beta h-EP showed a higher inhibitory activity than the parent peptide, [Tyr31]-analog had the same potency as beta h-EP, while [Trp27]-analog retained only one fourth the potency of beta h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted beta h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [3H]-dihydromorphine as the primary ligand showed that [Gln8]-analog had a binding potency 1.5-fold that of beta h-EP, while the potencies of [Tyr31]- and [Trp27]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their mu-receptor binding or analgesic activity. However, the relative potencies of binding to mu-receptor in [Gln8]- and [Tyr31]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to beta-EP receptor of all analogs appeared to be related to the charge properties of beta-EP molecule.

Animals↗

PubMed1987

Effects of equinatoxin on the guinea-pig atrium.

Equinatoxin is a lethal protein isolated from a sea anemone, Actinia equina. The toxin (0.1-3 micrograms/ml) caused an initial inhibition followed by an augmentation of contractions and beating rates in the isolated guinea-pig atrium. The inhibitory phase was transient (30-60 sec), while the stimulant phase lasted for about 30 min. The treated atrium showed tachyphylaxis to the toxin. The inhibitory effect of the toxin was diminished by tetrodotoxin and atropine and abolished by 8-phenyltheophylline or mepacrine. Dipyridamole, which blocks the uptake of adenosine in the heart, enhanced the inhibitory effect. The stimulant effect of the toxin was inhibited by indomethacin or mepacrine and abolished by a combination of both, but was not inhibited by propranolol. Bioluminescent assay performed during the inhibitory phase showed an increased release of ATP and radioimmunoassay during the stimulant phase revealed an increased release of prostaglandin E2 from the treated atrium. These results suggest that the cardiac inhibitory effect of equinatoxin is mainly due to release of adenyl compounds, while the cardiac stimulant effect of the toxin may result from the liberation of arachidonic acid and subsequent formation of prostaglandins in the guinea-pig atrium.

Adenosine Triphosphate↗

PubMed1987

Beta-endorphin: peripheral opioid activity of homologues from six species.

The peripheral opioid activity of six homologous beta-endorphins (beta-EPs) were assayed on the guinea pig ileum and the vas deferens of the mouse, the rat and the rabbit. In the guinea pig ileum assay, human beta-EP (beta h-EP) was less potent than camel, turkey, and ostrich beta-EPs, of the same potency as equine beta-EP and more active than des-acetyl salmon beta-EP. In the rat vas deferens, mammalian beta-EPs showed higher activity than those from the bird and the fish, whereas in the mouse vas deferens assay, beta h-EP is more active than those from other species. In the rabbit vas deferens, however, all homologous beta-EPs show very weak activity. The relative potency of beta-EP homologues obtained from rat vas deferens assay is in good correlation with the analgesic potency, while the receptor binding activity does not correlate with any of the four bioassays, but appears to be related to the charge properties of the peptides.

Amino Acid Sequence↗

PubMed1986

Hornetin: the lethal protein of the hornet (Vespa flavitarsus) venom.

By gel permeation on a Fractogel TSK HW 50 column followed by ion-exchange chromatography on carboxymethylcellulose CM 52, a lethal protein, designated hornetin, was purified from the venom of Vespa flavitarsus. Hornetin is a highly basic protein (pI 10.2) with a molecular mass of about 32 kDa. Its amino acid composition is characterized by a high content of lysine, aspartic and glutamic acid, and is devoid of tryptophan and cysteine. The lack of cysteine in the molecule is distinct from other known vespid venom proteins of comparable size. The i.v. LD50 of the toxin is 0.42 microgram per g mouse. Assayed on the red blood cells of the mouse and guinea-pig as well as isolated nerve muscle preparations of the chick and mouse, hornetin showed direct hemolytic activity and presynaptic neurotoxicity at microgram level and displayed musculotropic effect at higher concentrations.

Acetylcholine↗

PubMed1986

Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA.

The interaction of adenovirus-2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene. Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt-sensitive activities after associating with equal efficiency to both single- and double-stranded DNA; however, a putative C-terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti-serum specific to the EIA carboxy-terminus) showed 10-fold greater affinity to double- versus single-stranded DNA. When examined with Ad2 DNA, the EIA protein had a retention that was at least 2-fold higher compared to calf thymus DNA, suggesting some substrate specificity. It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III. This suggests that the strength of the protein interaction depends on the target DNA sequence. Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA-like protein potentiated the association of the protein to double-stranded calf thymus DNA up to 7-fold. These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co-factor(s) in an indirect fashion.

Adenovirus Early Proteins↗

PubMed1986

T-cell receptor variable region gene usage in T-cell populations.

We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens.

Animals↗

PubMed1986

Mechanisms of the inhibition by neostigmine of tetanic contraction in the mouse diaphragm.

Neostigmine (0.5-2 microM) caused fade of tetanic contractions (Wedensky inhibition) evoked by repetitive nerve stimulation. The mechanism underlying this action was studied in intact and cut isolated phrenic nerve-diaphragm preparations of mice. The fade was brought about by failure to elicit muscle action potentials. During fade, the muscle was unable to conduct directly evoked action potentials across the central endplate zone. Recovery of excitability occurred in 5 s with continued stimulation. In the presence of neostigmine, the resting membrane potential at endplate areas during repetitive stimulation decreased from -80 mV to less than -50 mV within the first 10 pulses at 75-200 Hz and thereafter recovered gradually to about -60 mV in the following 5 s during continuous stimulation. The quantal content of endplate potentials evoked by single stimulation was not reduced by neostigmine whereas that evoked by high frequency stimuli (75 Hz) was reduced to about 1/3 in 10 pulses. It is concluded that the fade of tetanic contraction caused by inhibition of acetylcholinesterase is induced by the inactivation of sodium channels in the area surrounding the endplates and that the sustained fade is due to a decrease of transmitter release. Both effects are the result of acetylcholine accumulation.

Action Potentials↗

PubMed1986

Differences in pharmacological actions between beta-bungarotoxin and other neurotoxic phospholipases A2 purified from snake venoms.

Five highly toxic phospholipases A2 (PLAs) (beta-bungarotoxin, caudoxin, Mojave toxin, notexin and a basic PLA from Naja nigricollis venom) were compared for their pharmacological actions. In the chick biventer cervicis nerve-muscle preparation, all PLA toxins except beta-bungarotoxin (beta-BuTX) inhibited the postsynaptic acetylcholine response and induced contracture of the muscle at a high concentration. Indirect hemolytic activity was found in all PLA toxins and some of the toxins (Naja nigricollis basic PLA and Mojave toxin) even showed a potent direct hemolytic action, while beta-BuTX was devoid of both direct and indirect hemolytic activities on the guinea-pig erythrocytes. All PLA toxins except beta-BuTX caused an increase in muscle tone in the guinea-pig ileum at a concentration as low as 0.05 microgram/ml, and an increase in the contractile force in the guinea-pig atrium at a concentration of 1.0 microgram/ml. In contrast, beta-BuTX had no stimulant effect at concentrations up to 10 micrograms/ml. On the cultured cells, beta-BuTX suppressed the proliferation of neuroblastoma cells, but did not cause lysis of non-neuronal cells of the rat brain. However, beta-BuTX uniquely maintained a high population of viable cells in the neuroblastoma cell cultures. From these results it was concluded that beta-BuTX is the most specific presynaptic neurotoxin among the PLA toxins so far tested.

Animals↗

PubMed1984

Plasmid-directed synthesis of genuine adenovirus 2 early-region 1A and 1B proteins in Escherichia coli.

The transforming region of human adenovirus 2 is located in the left 11.2% of the viral genome and is comprised of two distinct genetic units termed E1A and E1B. cDNAs containing the entire nucleotide sequence of the mature E1A 13S and E1B 22S mRNAs that are complementary to these genetic units have been introduced into bacterial plasmids a short distance downstream from the Escherichia coli lac promoter. Upon transformation into appropriate E. coli hosts, one of these plasmids, pKHAO, directed the synthesis of a 45-kilodalton (kd) protein, and the other, pKHBO, synthesized a protein of 54.9 kd. Both of these plasmid-encoded proteins constituted 0.1 to 0.3% of the total cellular protein and were virtually identical to the authentic adenovirus 2 E1A 42- to 50-kd and E1B 53- to 58-kd tumor antigens (T antigen) as determined by gel electrophoresis, immunoprecipitation, and tryptic fingerprint analysis. With the use of our pKHBO expression plasmid we were also able to demonstrate that the second AUG sequence appearing in the E1B 22S mRNA corresponded to the start of the gene encoding the large adenovirus 2 T antigen. This confirms theoretical deductions based on DNA sequencing analysis that translation of the large T antigen initiates translation at an internal ATG rather than at the 5'-proximal AUG.

Adenoviruses, Human↗

PubMed1982

[A new human hepatoma cell line: establishment and characterization].

We have grown a human hepatoma cell line, designated as HA22T/VGH, from a 52-yr-old male hepatoma patient since July 1, 1980. This cell line has been subcultured more than 100 passages. The chromosome analysis of HA22T/VGH indicated that the chromosome numbers varied from 70 to 146, with the mode of 73. Methylcellulose soft agar assay showed that approximately 40% of the HA22T/VGH cells formed colonies. The HA22T/VGH produced tumors in nude mice. Histopathological studies of the tumor revealed the arrangement of hepatoma. Detected by the complement fixation method HA22T/VGH cells secreted ceruloplasmin, Factor B, C3, C4, Gc-globulin and alpha 1-acid-glycoprotein. These cells contained the liver associated enzymes: alanine amino transferase, tyrosine amino transferase and gamma-glutamyl transferase. HBsAg and alpha-fetoprotein were not detectable in the HA22T/VGH culture media or cell lysates by the radioimmunoassay.

Animals↗