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Biomedical subjects

K Lin

Publications and source records attributed to K Lin.

At least 127 records · Page 7Linked to original sources
PubMed1991

Expression and site-directed mutagenesis of hepatic glucokinase.

Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli. Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data. The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP. The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family.

Animals↗
PubMed1991

Establishment of human myeloma cell line KM-2R and its preliminary application to human-human hybridoma research.

The authors report the establishment of a human myeloma cell line--KM-2R--and its preliminary use in human-human hybridoma research. KM-2R cells are resistant to both 6-TG and ouabain. Their doubling time is about 30 h. They have a modal chromosome number of 81-85. Using ELISA and immunodiffusion techniques, gamma-heavy and kappa-light chains were detected in the concentrated cell culture supernatant. KM-2R cells are sensitive to HAT medium. When fusing with normal human spleen cells, tonsil cells, and B lymphoblasts transformed by EB virus, fusion frequencies of 20-60% resulted.

Cell Division↗
PubMed1990

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis.

The current model for hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase divides the protein into two functional domains: an N-terminal kinase domain and a carboxyl-terminal bisphosphatase domain. Site-directed mutagenesis was used to evaluate the role of two putative bisphosphatase active site histidyl residues in catalysis. His-258 has been implicated as a phosphoacceptor (Pilkis, S. J., Lively, M. O., and El-Maghrabi, M. R. (1987) J. Biol. Chem. 262, 12672-12675), and the importance of this residue was confirmed when it was mutated to alanine and neither bisphosphatase activity nor a phosphoenzyme intermediate could be detected. Mutation of His-392 to alanine produced an enzyme which had five percent of wild-type fructose 2,6-bisphosphatase activity, and the rate of phosphoenzyme formations was decreased from 4800 nmol/min/mg to 2.9 nmol/min/mg. Mutation of His-392 to phenylalanine, lysine, or aspartic acid also produced proteins that did not hydrolyze fructose 2,6-bisphosphate or form a phosphoenzyme intermediate. These results are consistent with an important role for His-392 in the bisphosphatase reaction, probably as a proton donor, and with its designation as an active site residue based on homology modeling (Bazan (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). H258A had the same Vmax for 6-phosphofructo-2-kinase as the wild-type enzyme, and the mutant's kinase was inhibited by cAMP-dependent phosphorylation. In addition, H392F and H392K did not catalyze the kinase reaction, although H392D had normal kinase activity which was also modulated by cAMP-dependent phosphorylation in the same manner as the wild-type enzyme. Thus, an active bisphosphatase domain is not a necessary condition for phosphorylation-induced changes in 6-phosphofructo-2-kinase activity. The results also suggest that structural and/or active site interactions exist between the two domains of the enzyme.

Amino Acid Sequence↗
PubMed1990

Expression of mammalian liver glycolytic/gluconeogenic enzymes in Escherichia coli: recovery of active enzyme is strain and temperature dependent.

A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

[The specific photodynamic effect of monoclonal antibodies conjugated with hematoporphyrin derivative on gastric cancer in vitro and in vivo].

A murine monoclonal antibody (MoAb) BB4.3 raised against human gastric cancer cell line BGC823 was purified with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. The MoAb BB 4.3 was conjugated with hematoporphyrin derivative (HPD) by using carbodiimide. The conjugate HPD-BB4.3 retained both antibody activity and photochemical activity of HPD. In vitro the phototoxic effect of HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The target-selective photocytotoxicity of HPD-BB 4.3 was verified by the fact that this conjugate showed greater cytotoxicity than the corresponding normal mouse IgG (NIgG) conjugated with HPD, and that it showed less cytotoxicity to BB4.3 negative reaction cell line B-80 than to BGC 823; and moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody. In an in vivo study, nude mice inoculated with 2 X 10(5) BGC823 cells were administered HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS respectively; and were then exposed to light. Out of six animals treated with HPD-BB4.3 conjugate, four remained tumor-free for a long time. There was significant difference between HPD-BB 4.3 treated group and all control groups. The HPD-BB4.3 conjugate was also shown more than 40% inhibition of growth of established tumors in comparison with the control groups.

Animals↗
PubMed1987

[Study on serum TSH and its response to TRH measured by a high sensitive immunoradiometric assay during and after pregnancy].

With a new highly sensitive immunoradiometric assay (IRMA), serum thyrotropin (TSH) concentrations were determined in 213 healthy pregnant women. Serum free thyroxine (FT4) and human chorionic gonadotropin (HCG) levels were also measured in the same individuals. The mean serum TSH value in the 1st trimester was 0.8 microU/ml and significantly lower than those of the other three periods and non-pregnant women. Seventeen of 77 (22.1%) and 2 of 128 (1.6%) subjects in the 1st and 3rd trimester, respectively, had an undetectable serum TSH value (less than 0.1 microU/ml) with normal or slightly elevated serum FT4 levels and these pregnant women had no clinical signs or symptoms of thyrotoxicosis. Significant positive correlations were found between serum FT4 and HCG in the 1st and 3rd trimester. In 13 subjects whose serum basal TSH values had been less than 0.1 microU/ml, the thyrotropin-releasing hormone (TRH) stimulation test was performed. Two of 4 subjects in the 1st trimester and one of 8 subjects in the 2nd trimester did not respond to TRH and their serum TSH values before TRH administration were less than 0.1 microU/ml. Although the exact mechanism of blunted TSH response to TRH is not clear, the feedback suppression of serum TSH by slightly elevated serum FT4 concentrations may occur early in pregnancy. However, in the 3rd trimester serum FT4 values fell below the reference range without an elevation of serum TSH. Other factors regulating the secretion of TSH during pregnancy can be postulated.

Female↗
PubMed1985

Tay-Sachs disease associated with precocious puberty.

A girl with Tay-Sachs disease developed enlargement of the mammary glands at the age of 4 years and menstruation at the age of 6 years. It was demonstrated that the precocious puberty in this patient was due to the hypersecretion of gonadotropins.

Child↗
PubMed1983

Implications of a 5'-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer.

5'-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56 degrees C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37 degrees C for 1 h and it was destroyed completely by heating at 100 degrees C for 30 min. When the heated (56 degrees C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of N-acetyl-beta-D-glucosaminidase or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5'-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35,000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggest that the previously reported undetectability of 5'-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5'-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5'-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.

5'-Nucleotidase↗
PubMed1983

Scintigraphic demonstration of tracheo-esophageal fistula.

A tracheo-esophageal fistula, developed following radiotherapy for an esophageal carcinoma, was vividly demonstrated by radionuclide imaging. The abnormality was later confirmed by a barium esophagram and endoscopic examinations. The scintigraphic procedure, making use of a Tc-99m sulfur colloid swallow, appears to be a simple alternative method that may be clinically useful for the diagnosis of such a condition.

Barium Sulfate↗
PubMed1969

Some properties of the microsomal 2,3-oxidosqualene sterol cyclase.

The transformation of 2,3-oxidosqualene to lanosterol is catalyzed by a microsomal enzyme (cyclase) which can be obtained in soluble and partially purified form by treatment of liver microsomes with deoxycholate as previously shown. The catalytic and physical properties of the soluble enzyme are determined by ionic strength. In 0.4 M KCl the cyclase exists largely in a dissociated, enzymatically active form. Solutions of low ionic strength (0.1 M KCl or less) cause enzyme aggregation and loss of activity. The anionic detergent deoxycholate is essential for cyclase activity, but is effective only in a narrow concentration range.

Animals↗