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Biomedical subjects

Y T Kim

Publications and source records attributed to Y T Kim.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein.

Bacteriophage T7 gene 2.5 protein has been purified to homogeneity from cells overexpressing its gene. Native gene 2.5 protein consists of a dimer of two identical subunits of molecular weight 25,562. Gene 2.5 protein binds specifically to single-stranded DNA with a stoichiometry of approximately 7 nucleotides bound per monomer of gene 2.5 protein; binding appears to be noncooperative. Electron microscopic analysis shows that gene 2.5 protein is able to disrupt the secondary structure of single-stranded DNA. The single-stranded DNA is extended into a chain of gene 2.5 protein dimers bound along the DNA. In fluorescence quenching and nitrocellulose filter binding assays, the binding constants of gene 2.5 protein to single-stranded DNA are 1.2 x 10(6) M-1 and 3.8 x 10(6) M-1, respectively. Escherichia coli single-stranded DNA-binding protein and phage T4 gene 32 protein bind to single-stranded DNA more tightly by a factor of 25. Fluorescence spectroscopy suggests that tyrosine residue(s), but not tryptophan residues, on gene 2.5 protein interacts with single-stranded DNA.

Adenosine Triphosphate↗
PubMed1992

Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.

Base Sequence↗
PubMed1992

Radiation pneumotoxicity in rats: modification by inhibitors of angiotensin converting enzyme.

The present study determined whether inhibitors of angiotensin converting enzyme (ACE) can ameliorate radiation-induced pulmonary endothelial dysfunction and pulmonary fibrosis in rats sacrificed 2 months after a range of single doses of 60Co gamma rays to the right hemithorax. Four indices of pulmonary endothelial function were monitored: right lung ACE and plasminogen activator (PLA) activity, and prostacyclin (PGI2) and thromboxane (TXA2) production. Hydroxyproline (HP) content served as an index of pulmonary fibrosis. Rats consumed either control powdered chow or feed containing one of five modifying agents continuously after irradiation. The modifiers included three ACE inhibitors: Captopril, CL242817, and CGS13945, respectively, a thiol, a thioacetate, and a nonthiol compound. All of the ACE inhibitors are analogues of proline. Two additional modifiers were tested: penicillamine, a thiol with no ACE inhibitory activity; and pentoxifylline, a vasodilator that is neither a thiol nor an ACE inhibitor. Radiation produced a dose-dependent decrease in lung ACE and PLA activity, and an increase in PGI2 and TXA2 production and in HP content. All ACE inhibitors attenuated the radiation-induced suppression in lung ACE and PLA activity. All thiol or thioacetate compounds ameliorated the radiation-induced increase in PGI2, TXA2, and HP. The two agents that were both thiols and ACE inhibitors (Captopril and CL242817) spared all of the radiation reactions, while the compound that was neither a thiol nor an ACE inhibitor (pentoxifylline) spared none of the reactions. These data suggest a novel application for ACE inhibitors in general, and for Captopril in particular, as modifiers of radiation pneumotoxicity.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1992

MRI classification of lumbar herniated intervertebral disc.

A consecutive series of 28 patients with lumbar herniated intervertebral disc (HIVD) who exhibited positive magnetic resonance imaging (MRI) findings was analyzed and compared with operative findings to define the type of disc herniation. This prospective study showed that the overall accuracy of MRI in predicting the types of HIVD was 80.6%. Proton density image was the most helpful in differentiating protruded discs from extruded ones. Disruption of "posterior longitudinal ligament line," defined on T2-weighted sagittal image, was reliable in differentiating a transligamentous disc from subligamentous one. Sequestered disc could be more accurately diagnosed with Gadolinium-DTPA enhancement, which showed anterior rim enhancement of the sequestered portion on T1-weighted axial image.

Adolescent↗
PubMed1992

Pentoxifylline does not spare acute radiation reactions in rat lung and skin.

Male rats were exposed to single doses (0-30 Gy) of 60Co gamma rays to the right hemithorax. Half of each dose group consumed only control powdered chow after irradiation, and half consumed feed containing 0.10% (w/w) pentoxifylline (50 mg/kg/day). The severity of epilation and desquamation in the field of the radiation port was scored weekly. Two months after irradiation the animals were killed, and pulmonary endothelial function was monitored by the activity of lung angiotensin converting enzyme (ACE) and plasminogen activator (PLA), and by production of prostacyclin (PGI2) and thromboxane (TXA2). The amount of hydroxyproline (HP) in the lung served as an index of pulmonary fibrosis. Radiation produced a dose-dependent decrease in ACE and PLA activity in the right lung and an increase in the production of PGI2 and TXA2. This endothelial dysfunction was accompanied by an increase in wet weight and in protein and HP content in the irradiated lung. Pentoxifylline spared only the increase in lung wet weight and protein content, and actually elevated the radiation-induced hyperproduction of PGI2 and TXA2. The severity of the epilation and desquamation reactions increased with increasing radiation dose and time but was independent of diet. These data indicate that pentoxifylline, despite some promising pharmacological actions, has no beneficial effect on acute radiation reactions in rat lung and skin.

Administration, Oral↗
PubMed1991

4-Aminobutyrate aminotransferase: identification of lysine residues connected with catalytic activity.

Bis-PLP (P'P2-bis[5'-pyridoxal]diphosphate) was used as a probe of the catalytic site of 4-aminobutyrate aminotransferase. It reacts with lysine residues connected with aminotransferase activity and the binding of 1 mol of reduced bis-PLP/enzyme monomer abrogates catalytic activity. The reactive lysine residues are characterized by low pK values (pK = 7.3). The presence of substrate 2-oxoglutarate (4 mM) prevents inactivation of the aminotransferase treated with bis-PLP. After tryptic digestion of the enzyme modified with bis-PLP and reduced with tritiated NaBH4, a radioactive peptide absorbing at 320 nm was separated by reverse-phase high-performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysine residue of monomeric 4-aminobutyrate aminotransferase has reacted with bis-PLP. The sequence of the modified peptide differs from the sequence of the peptide bearing the cofactor pyridoxal-5-P covalently attached to a lysine residue. It is postulated that the modified lysine residue is involved in direct interactions with negatively charged carboxylic groups of 2-oxoglutarate.

4-Aminobutyrate Transaminase↗
PubMed1991

Distinctions in growth cone morphology and motility between monopolar and multipolar neurons in Drosophila CNS cultures.

Growth cones play a central role in determining neurite extension, pathfinding and branching, and in establishing synaptic connections. This paper describes an initial characterization of growth cone morphology and behavior in dissociated larval central nervous system (CNS) cultures of Drosophila. Contrast-enhanced video images of growth cones in monopolar and multipolar neurons were characterized by employing morphometric parameters such as the number and length of filopodia, and the area and roundness of the lamellipodia. Behavior of growth cones was analyzed by a motility index and boundary flow plots originally devised for measuring motility in other cellular systems. We found that separate CNS regions yielded cultures of different major cell types with distinct neuritic patterns that could be correlated with the morphology and motility of the associated growth cones. Monopolar neurons were the major cell type in brain cultures, whereas multipolar neurons were predominant in ventral ganglion cultures. Moreover, the growth cones of monopolar neurons, which are likely to be associated with the axonal processes, differed from those of multipolar neurons, which might be related to dendritic terminals. Growth cones in monopolar neurons had larger lamellipodia of less erratic shape accompanied by fewer and shorter filopodia, and, when active, displayed much higher motility and less directionality in motion. Alternatively, these morphological and behavioral distinctions between monopolar and multipolar neurons may result from intrinsic differences in membrane adhesion and intracellular transport properties.

Animals↗
PubMed1991

"Cross-wiring" of the immune response in old mice: increased autoantibody response despite reduced antibody response to nominal antigen.

Older humans and experimental animals have been repeatedly found to have higher titers of autoantibodies than do younger individuals despite the impaired responses of older individuals to foreign antigens. The studies reported here were designed to examine the relationship between these two age-related changes in antibody responses. Antibody response to foreign antigen was measured concurrently with autoantibody response in the same mice. Old mice (18-24 months old) had decreased responses to foreign antigens and increased responses to bromelain-treated syngeneic erythrocytes, compared to young mice (2 months old). In vitro mixing experiments were consistent with the possibility that suppressor cell activity in spleen cells from old mice reduce the antibody response to foreign antigen but not to autologous antigen. The results support an emerging view that age-associated changes in immune responses are the result of dysregulation rather than exhaustion of the immune system.

Aging↗
PubMed1991

Role of induction chemotherapy in invasive cervical cancer.

The 386 cases of invasive cervical carcinoma treated with radiotherapy alone were statistically analyzed to delineate the high risk factors (HRFs) associated with a significantly high treatment failure rate; they were (1) stages III-IV, (2) lesion greater than or equal to 4.0 cm, (3) small cell carcinoma or adenocarcinoma, (4) stages I-II with lesion greater than or equal to 4.0 cm, and (5) lymphographic evidence of nodal metastasis. Then, chemoradiotherapy (induction chemotherapy plus subsequent radiotherapy) was instituted to 113 invasive cervical carcinoma patients with at least one such HRF. Each patient received two to three cycles of induction chemotherapy at about 3-week intervals. For squamous cell carcinoma, cisplatin, 100 mg/m2 iv, was followed immediately by 5-fluorouracil, 1000 mg/m2, as a 24-hr iv infusion x 5 days. For adenocarcinoma, cisplatin, 70 mg/m2 iv, on Day 1 was followed by cytoxan, 250 mg/m2, on Day 2, and adriamycin, 45 mg/m2, on Day 3. Five-year survival of these patients according to each HRF, in the above order, was 69.1, 67.2, 68.1, 78.3, and 79.5% after chemoradiotherapy, all significantly higher than 57.4, 53.0, 54.5, 48.0, and 48.8% by radiotherapy alone. Drug toxicities such as leukopenia, hepatotoxicity, nephrotoxicity, and hypomagnesemia were seen in 46.5, 53.2, 47.1, and 55.4% of all cycles, respectively. The toxicities altered drug schedule in 191 (61.2%) ongoing induction chemotherapy cycles. Our cisplatin-based induction chemotherapy is considered an effective preradiotherapy adjunct that can reduce treatment failure in HRF-associated invasive cervical carcinoma.

Adenocarcinoma↗
PubMed1991

The efficacy of script contexts in language comprehension intervention with children who have mental retardation.

This study was designed to investigate the effects of script-based and nonscript-based treatment on the language comprehension of 4 preschool children with mental retardation. An alternating treatments design (ATD) in combination with a multiple baseline design was used to evaluate treatment effects. Treatments were comprised of three script routines and three nonscript activities. Two semantic constructions were selected for training and counterbalanced across subjects and treatments. Daily probes were administered to assess the effects of the treatments. The script-based treatment was more effective than the nonscript treatment in facilitating comprehension of the targeted semantic constructions in 3 of the 4 subjects.

Adolescent↗
PubMed1990

Cellular basis for the age-associated increase in autoimmune reactions.

The mechanisms that lead to the increased expression of autoantibodies with age are poorly understood. We have studied the number, size, and density of spleen and peritoneal cells from young and old BALB/c and C57BL/6 mice as well as the frequency of clonal precursors for antibodies to mouse erythrocytes, thyroglobulin, and IgG in these lymphoid preparations. Old mice have a 6-fold increase in the number of resident peritoneal cells and a 2-fold increase in the absolute number of Ly1-bearing B cells in this population. Furthermore, old mice have twice as many large, low density splenic B cells as young mice. The frequencies of B cell clonal precursors for anti-BrMRBC and anti-thyroglobulin antibody-forming cells in old mice were 3-10 times greater than in young mice. In the same cultures, however, no increase in the frequencies of B cell clonal precursors for anti-IgG or anti-DNA antibody forming cells was detected in old compared to young mice. These findings and other data suggest that there are at least two families of B cell autoantibody precursors, one including anti-BrMRBC and anti-thyroglobulin autoantibodies, the other including anti-IgG and anti-DNA antibodies. Studies of the differential regulation of these two families of autoantibody precursors might contribute to a greater understanding of autoimmune phenomena in age and disease.

Aging↗
PubMed1990

Pyridoxal kinase. Structure and function.

Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by HPLC and used for binding studies with ATP and pyridoxal analogues. The spectroscopic properties of trinitrophenyl-ATP bound to the 24-kDa fragment are indistinguishable from those of TNP-ATP bound to the native kinase. The small 16-kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the analogues. The same pattern of digestion was observed when IAF pyridoxal kinase, carrying a fluorescent probe covalently bound to a specific SH residue, was preincubated with chymotrypsin. The kinetics of proteolysis of IAF-pyridoxal kinase was monitored by emission anisotropy, and the analysis of the initial rate of proteolysis at various concentrations of chymotrypsin reveals that the rate of unfolding of native pyridoxal kinase plays a dominant role in the proteolytic process.

Adenosine Triphosphate↗
PubMed1990

Allelic interactions at the shibire locus of Drosophila: effects on behavior.

This report describes a detailed behavioral study of four shi alleles, shits1, shits2, shits4, and shiST139, and their heteroallelic combinations. Flies of different heteroallelic combinations (shi/shi) were less sensitive to high temperature than the corresponding homozygotes. Among them, shits2/shits4 showed a striking reduction in temperature sensitivity. In contrast, different shi/+ heterozygotes were more severe in temperature sensitivity than the hemizygotes, Df/+. The hemizygous combinations of shi alleles over deficiency (shi/Df) were not completely lethal, with shits2 conferring distinctively higher and shits4 lower viability. A novel behavior, bang sensitivity, was also found in shi/Df. The results of allelic interactions suggest that the shi mutations examined appear to be antimorphic and that the shi gene products are likely to function in multimeric form.

Alleles↗
PubMed1990

Reversible inhibition of endocytosis in cultured neurons from the Drosophila temperature-sensitive mutant shibirets1.

The Drosophila mutant, shibirets1 (shits1), is paralyzed at restrictive temperatures (greater than 29 degrees C) by a reversible block in synaptic transmission. Heat pulses deplete synaptic vesicles in nerve terminals and inhibit endocytic internalization of plasma membrane in garland cells and oocytes. In dissociated cultures of larval central nervous system (CNS), a temperature-sensitive defect is also expressed in shits1 neurons: at 30 degrees C, growth cone formation is retarded and neurite outgrowth is arrested. We now report that we have examined constitutive endocytosis in Drosophila CNS culture and have demonstrated directly an endocytic defect in shits1 neurons. At the permissive temperature, 20-22 degrees C, both shits1 and wild-type neurons actively endocytosed fluorescein-labelled dextran (40 KD, 5%) or horseradish peroxidase (HRP, 1%). Within 5 min, HRP was seen in vesicles, cup-shaped bodies, tubules and multivesicular bodies in neurites and cell bodies. In contrast, endocytosis was inhibited in cultures derived from the temperature-sensitive paralytic shits1 by a 15 min heat pulse (30 degrees C). Even after 30 min of HRP exposure at 30 degrees C, HRP-containing membranes were absent from almost all shits1 neurites; a minority of cell bodies had a few HRP-containing vesicles. The temperature-dependent block in endocytosis was readily reversed at 20 degrees C. Interestingly, the block was overcome by high concentration of external cations: shits1 neurons in culture actively took up HRP in numerous vesicles at 30 degrees C if 18 mM Ca2+ or Mg2+ was added to the medium. Our results support the notion that membrane recycling plays a critical role in regulating neurite outgrowth. This study also provides baseline information for further mutational analysis of the mechanism underlying the membrane cycling process in cultured neurons.

Animals↗
PubMed1990

Immunobiology of aging and cancer.

Although the incidence of neoplasms is increased in the elderly, some tumors appear to grow more slowly in old as compared to young patients. We have used the B16 melanoma to explore the relationship between age, T-lymphocyte function, and the rate of tumor growth. Increasing age is associated with a decreased rate of tumor growth and impaired T-cell function in C57BL/6 mice. Furthermore, when T-cell immunity in young mice is compromised by thymectomy, B16 tumor growth is decreased. Mixed cell transfer studies demonstrated that T cells from young but not old donors stimulate the growth of B16 melanoma cells in young lethally irradiated recipients. Recently, T cells from young but not old mice have been reported to produce angiogenic factors. As tumors from young mice have a richer vascular supply than in old mice, it appears that one mechanism for the age-associated decrease in B16 melanoma growth is the decreased capacity of T cells from old mice to generate angiogenic factors. However, parabiosis of young and old mice showed that local factors in old mice also limit the stimulatory influences of T cells from young mice. Novel therapeutic approaches to limit the growth of tumors might result from a greater understanding of the production of and response to angiogenic factors produced by T cells.

Aging↗
PubMed1989

Activation of a flavoprotein by proteolysis.

Chymotryptic digestion of brain pyridoxine-5-P oxidase brings about a 4-fold enhancement of the catalytic power (Vmax/KM) using pyridoxine-5-P as substrate in the assay mixtures. The chymotrypsin-treated enzyme is less susceptible to inhibition by pyridoxal-5-P than the native enzyme. Fragments arising from limited proteolysis were separated by affinity chromatography using P-pyridoxal-Sepharose as supporting matrix. Catalytically active fractions, eluted by pyridoxine-5-P (5mM), displayed three bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of the three protein bands are considerably lower than 28 kDa, the molecular mass of monomeric pyridoxine-5-P oxidase. Spectroscopic studies, absorption, fluorescence, and circular dichroism revealed that the microenvironment surrounding the cofactor flavin mononucleotide is not perturbed by limited proteolysis.

Animals↗
PubMed1989

Sequence of the cysteinyl-containing peptides of 4-aminobutyrate aminotransferase. Identification of sulfhydryl residues involved in intersubunit linkage.

Three cysteine-containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4-dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species. To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S-DABIA-labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides II, III) have been oxidized. The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.

4-Aminobutyrate Transaminase↗