Purification and characterization of recombinant melanoma growth stimulating activity.
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Amino Acid Sequence↗
Biomedical subjects
J H Han
Publications and source records attributed to J H Han.
Use of a signature nucleotide sequence of hepatitis C virus for detection of viral RNA in human serum and plasma.
The nucleic acid sequence of the putative 5'-untranslated (5PUT) region of hepatitis C virus (HCV), determined for samples obtained from a variety of geographic origins, was found to be over 98% conserved among all isolates. On the basis of this signature sequence for HCV, a viral RNA assay was developed by using cDNA synthesis with reverse transcriptase, followed by polymerase chain reaction (PCR). The new assay was compared with the Ortho-Chiron C100-3 HCV enzyme-linked immunosorbent assay to research radioimmunoassays for antibodies to the C33c and C22 HCV antigens and to the first reported set of HCV PCR primers designed from the NS3 domain. Plasma samples from 16 Japanese patients with non-A, non-B hepatitis (NANBH) and 16 immunoassay-positive blood donors from the United States were investigated. The 5PUT PCR primers were found to be superior to the NS3 primers in sensitivity and specificity (15 of 25 versus 3 of 25 of the C100 enzyme-linked immunosorbent assay-positive samples, respectively). Samples from two C100-negative patients with acute NANBH were found to react with the 5PUT primers but not with the NS3 primers. Also, two of three patients with chronic NANBH converted from reverse transcriptase PCR positive to negative after interferon treatment. Although the clinical significance of the presence or absence of HCV RNA in samples from patients is not fully understood, the use of probes and primers from the 5PUT region (as opposed to primers from other segments) should not lead to false-negative results due to nucleic acid sequence variations in viral isolates.
Base Sequence↗
Effect of ursodeoxycholic acid on experimental hepatic porphyria induced by griseofulvin.
Griseofulvin(GF) has become the drug of choice as an antifungal agent for patients who suffer from many kinds of fungal infection. In order to clarify hepatic injury by griseofulvin(GF) overload and the effect of UDCA on GF-induced hepatic injury, the authors carried out biochemical, histologic, and ultrastructural studies of liver following treatment with griseofulvin and ursodeoxycholic acid(UDCA) in mice. Urine porphobilinogen excretion in the group treated with GF alone was significantly increased and reached the highest level in the 4th week and declined thereafter. Biochemical studies of the liver function showed no remarkable changes of serum bilirubin levels throughout the experimental period in all groups, except for SGPT and alkaline phosphatase activities which were significantly elevated and reached the highest level in the second week. Then they slightly decreased in GF treated groups(GF alone and GF plus UDCA) in comparison with the control group. Pathologic findings in the group treated with GF alone include focal liver cell necrosis(esp, zone 3), Mallory bodies in hepatocytes(esp, zone 1), Kupffer cell activation, and brown protoporphyrin pigments in the hepatocytes, bile canaliculi and interlobular bile ducts with a marked inflammatory cell infiltration in the portal tracts. Under the polarizing light microscope, bile ductular and canalicular thrombi showed a "Maltese cross" birefringence in mice treated with GF alone. There is no definite finding of fatty change in hepatocyte. Under the microscope, the liver appeared normal with an intact lobular architecture in the GF plus UDCA treated group. Electron microscopically, GF-induced changes include swelling of mitochondria, globular protoporphyrin crystals in the hepatocyte cytoplasm, markedly dilated bile cannaliculi and bile ducts and the formation of a Mallory hyaline bodies in the hepatocytes. There were no noticeable structural changes in the GF plus UDCA-treated group. Therefore the results suggest that GF causes hepatic injury, namely porphyria and cholestasis, and the treatment of UDCA may have cytoprotective and choleretic effects on GF-induced hepatic injuries.
Alanine Transaminase↗
Recombinant expression, biochemical characterization, and biological activities of the human MGSA/gro protein.
Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterization shows that the recombinant protein is identical with the natural protein and is devoid of posttranslational glycosylation, sulfation, and phosphorylation. The two C-terminal amino acids are proteolytically removed from the mature recombinant MGSA, indicating a length of 71 instead of the predicted 73 amino acids. The recombinant MGSA is mitogenically active on the Hs294T melanoma cells. The purified MGSA competes with interleukin 8 for binding to neutrophil receptors and exhibits neutrophil chemotactic activity equivalent to that of interleukin 8.
Amino Acid Sequence↗
Purification and characterization of human plasminogen activator inhibitor type I expressed in Saccharomyces cerevisiae.
The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.
Antigens↗
Psychiatric epidemiology in Korea. Part I: Gender and age differences in Seoul.
The most dramatic finding was the very high prevalence of alcohol abuse, using DSM-III criteria, among men in Seoul, Korea. The prevalence of other psychiatric disorders was lower than in St. Louis, Missouri. With the current biological emphasis in psychiatry, questions may be raised regarding the different prevalence rates of schizophrenia, affective disorders, and even alcoholism. The deficit of the aldehyde dehydrogenase isoenzyme 1 has been hypothesized to reduce the prevalence of alcohol abuse among Asians. Twenty-five percent of Koreans have been found to be deficient in the enzyme, but despite this their prevalence of alcohol abuse is higher than among Americans. Cultural issues are paramount in the much lower prevalence of alcohol abuse among women in Korea.
Adolescent↗
Psychiatric epidemiology in Korea. Part II: Urban and rural differences.
This paper compares the findings in rural Korea and in the urban capital city of Seoul, Korea. In rural Korea 1966 subjects and in Seoul 3134 subjects were interviewed with the National Institute of Mental Health Diagnostic Interview Schedule. We found that the prevalence of psychiatric disorders, including alcohol dependence, was higher in rural Korea than in the urban setting. Comparing men and women, the prevalence of psychiatric disorders was higher in women. The exception was alcohol abuse/dependence, which was much higher among men, as was antisocial personality disorder. Comparison of prevalence by age groups showed that, in contrast to American studies, there was a tendency for prevalence to increase with increasing age. Alcohol abuse/dependence was much higher in rural Korea compared with St. Louis. Drug abuse/dependence was much higher in St. Louis. Other interesting similarities and differences are discussed.
Adolescent↗
[Intestinal parasite infections among inhabitants in two islands of Tongyeong-gun, Kyeongsangnam-do].
This study was performed to evaluate the status of intestinal parasitic infections among the inhabitants in two islands (Chu-do and Doomi-do) of Tongyeong-gun, Kyeongsangnam-do (Province), from August to September, 1989. A total of 189 stool specimens was collected from the inhabitants of 3 villages and examined by Kato's cellophane thick smear and formalin-ether sedimentation techniques. Stoll's dilution egg counting technique was done for the quantitative examination of helminth eggs. The overall positive rate of intestinal parasites was 30.2%. The egg positive rate of Ascaris lumbricoides was 2.1%, that of Trichuris trichiura 24.3%, hookworm 2.1%, Trichostrongylus orientalis 0.5%, Clonorchis sinensis 1.1%, heterophyid 1.6%, and Taenia species 2.6%. The cyst positive rate of Giardia lamblia was 1.6% and that of Entamoeba coli 0.5%. In T. trichiura infection, the egg positive rate of females (29.9%) was much higher than that of males (17.2%). Among the age groups, 10-19 year group showed the highest infection rate, 32.4%. It was revealed that the prevalence of intestinal parasitic infections among the inhibitants of remote islands should be still high in comparison with ever-reported ones in urban or rural areas.
Adolescent↗
Lipogenesis stimulatory and inhibitory activities of the insulin mediators.
Incubating plasma membranes prepared from pig liver with varying concentrations of insulin (50-1000 microU/ml) resulted in the release of at least two insulin chemical mediators. One of them was fraction 1 of insulin mediator (M. W. 3700-4000 daltons) which had a significant lipogenesis-stimulating activity. The other was fraction 2 of insulin mediator (M. W. about 1000 daltons) which exhibited a lipogenesis-inhibitory activity. The ratio of yield between the two mediators produced from the membranes was not only dependent on the concentration but also on the potency of insulin and its analogs added. The result showed that there was more production of fraction 2 than fraction 1 with the inducer at low concentration (100 microU/ml), while the production of fraction 1 from the plasma membranes incubated with high concentration of insulin (300 microU/ml) was higher than fraction 2. On the other hand, insulin and its analogs which have different biological activities and receptor binding activities have been used to induce the insulin mediators. The results obtained were similar to those mentioned above. This suggested that the generation of the mediators was dependent on the biological potences but not the binding activities.
Animals↗
Interleukin-1, endotoxin or tumor necrosis factor/cachectin enhance the level of plasminogen activator inhibitor messenger RNA in bovine aortic endothelial cells.
It is known that either endotoxin (LPS) or interleukin-1 (IL-1) increase the activity of plasminogen activator inhibitor (PAI) in the culture media of human and bovine endothelial cells. We have confirmed these results in bovine aortic endothelial cells (BAEC). To determine if this effect was mediated by increases in the level of PAI messenger RNA (mRNA) we examined the effects of these cytokines on PAI mRNA levels in BAEC, using RNA blot analyses. Treatment of BAEC with either IL-1, LPS, or human recombinant tumor necrosis factor/cachectin (TNF) dramatically increased the level of PAI mRNA. Since elevated levels of PAI will decrease fibrinolytic potential, this mechanism is in concert with the known increase in in vivo procoagulant potential induced by these agents and could contribute to thromboembolic phenomena.
Animals↗
Cloning and expression of cDNA encoding antistasin, a leech-derived protein having anti-coagulant and anti-metastatic properties.
As a factor Xa inhibitor, antistasin is a potent anti-coagulant and anti-metastatic agent that is found in the salivary gland of the Mexican leech Haementaria officinalis. cDNA clones that encode antistasin have been isolated. Subsequent sequence analysis and comparison with the amino acid sequence of the mature protein indicates that antistasin is produced as a pre-protein containing a 17-amino acid signal peptide. Antistasin exists as at least two variants. By sequence analysis of multiple cDNA clones, we found two additional sites for amino acid substitutions, confirming variants that differ from each other by amino acid changes at a minimum of four residues. These sequence variations appear to be the result of allelic variation rather than gene duplication as deduced from DNA blot analyses. Sequence data suggest that antistasin may have evolved from a smaller ancestral gene by a duplication event giving rise to a two-fold structural homology between the N- and C-terminal halves of the molecule. Insect cells transfected with a recombinant baculovirus expressed antistasin which was biologically active and had an electrophoretic mobility identical to that of the native molecule.
Amino Acid Sequence↗
Evolution of the genome and the genetic code: selection at the dinucleotide level by methylation and polyribonucleotide cleavage.
Noting the scarcity of CpG dinucleotide in total genomic DNA derived from higher organisms and the scarcity of TpA dinucleotide in total genomic DNA derived from most life forms, we examined the distribution of these dinucleotides in sequences derived from functionally distinct types of human DNA, including mitochondrial DNA, intergenic DNA, intron DNA, and DNA destined to be represented in the cytoplasm as mRNA, tRNA, or rRNA. While CpG frequency has fallen to its lowest levels in DNA that is transcriptionally silent, TpA is most stringently excluded in DNA destined to be expressed as mRNA in the cytosol. This observation suggests that the selective pressures leading to the removal of CpG and TpA operate at different levels. With respect to TpA, dinucleotide scarcity may reflect a requirement for mRNA stability and may indicate the action of UpA-selective ribonucleases. We propose that, by reason of its instability, UpA must have been very rare in primordial RNA. Therefore, tRNA with the anticodon for this dinucleotide may have failed to evolve, making UpA the primordial doublet "stop" codon. The modern triplet code has faithfully conserved this arrangement in the two universal stop codons, UAA and UAG.
Base Sequence↗
Lambda gt22S, a phage expression vector for the directional cloning of cDNA by the use of a single restriction enzyme SfiI.
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Bacteriophage lambda↗
[Cloning and location of the penicillin G acylase gene from E. coli AS1.76].
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Amidohydrolases↗
Adenylate cyclase-modulating mediators of insulin.
Plasma membranes prepared from pig liver incubated with insulin (50-300 microU/ml) resulted in the release of at least two insulin chemical mediators. They appeared to modulate the activity of adenylate cyclase in liver plasma membranes of pig. One of them was fraction 1 of insulin mediator (M. W. about 3700-4000 dalton) which markedly stimulated the activity of the enzyme, the other was fraction 2 of insulin mediator (M. W. about 1000 dalton) which inhibited the enzyme activity. The results showed that the inhibitor of fraction 2 generated was significantly higher than that of fraction 1 when the membranes were incubated with insulin of low concentration (50-100 microU/ml). On the other hand, the generation of stimulator of fraction 1 from plasma membranes incubated with insulin of high concentration (200 microU/ml) was higher than that of fraction 2. So the ratio of yield between two mediators produced from the membranes was dependent on the concentration of insulin added. The results also showed that the effect of fraction 1 of insulin mediator on adenylate cyclase activity in liver cell plasma membranes was biphasic while fraction 2 of insulin mediator showed an inhibitory effect only even though it was at very high concentration. The results that both mediators combined with Gpp(NH)p and forskolin to affect the enzyme activity show that the action of insulin mediators likely resides in the GTP regulatory component of adenylate cyclase.
Adenylyl Cyclases↗
Lambda gt22, an improved lambda vector for the directional cloning of full-length cDNA.
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Bacteriophage lambda↗
Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning.
We have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase [Van den Bosch, H., Aarsman, A. J., DeJong, G. N., & Van Deenen, L. M. (1973) Biochim. Biophys. Acta 296, 94-104].
Amino Acid Sequence↗
The nationwide epidemiological study of mental disorders in korea.
The lifetime prevalences of DSM-III mental disorders using Korean version of DIS-III are presented. They were studied in 5,100 adults (aged 18 to 65) in household selected by two stage cluster sampling. Comparisons were made between regions, sex and age groups. International comparison with Epidemiologic Catchment Area program was also made.
Adolescent↗