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J Lin

Publications and source records attributed to J Lin.

At least 55 records · Page 3Linked to original sources
PubMed1989

Determination of the roles of active sites in F1-ATPase by controlled affinity labeling.

The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl) [alpha-32P]ATP (FDNP-[alpha-32P]ATP) and 3'-O-(5-fluoro-2,4-dinitrophenyl) [8-14C]ATP (FDNP-[14C]ATP) were synthesized and used to characterize the structure and function of the three active sites in F1-ATPase. FDNP-[alpha-32P]ATP was found to bind covalently to F1 up to two DNP-[alpha-32P]ATP labels per F1 in the absence of Mg2+ without decreasing the ATPase activity. However, when MgCl2 was subsequently added to the reaction mixture, the enzyme could be further labeled with concomitant decrease in ATPase activity that is consistent with the complete inactivation of one enzyme molecule by an affinity label at the third ATP-binding site. Partial hydrolysis of the FDNP-[14C]ATP-labeled enzyme and sequencing of the isolated peptide indicated that the affinity label was attached to Lys-beta 301 at all three active sites. Samples of F1 with covalent affinity label on Lys-beta 301 were also used to reconstitute F1-deficient submitochondrial particles. The reconstituted particles were assayed for ATPase and oxidative phosphorylation activities. These results show that the catalytic hydrolysis of ATP either by F1 in solution or by F0F1 complex attached to inner mitochondrial membrane takes place essentially at only one active site, but is promoted by the binding of ATP at the other two active sites, and that ATP synthesis during oxidative phosphorylation takes place at all three active sites [corrected].

Adenosine Triphosphatases↗
PubMed1989

8-Azido-2'-O-dansyl-ATP. A fluorescent photoaffinity reagent for ATP-binding proteins and its application to adenylate kinase.

The photoaffinity reagent 8-azido-2'-O-[14C]dansyl-ATP (AD-ATP) has been synthesized for labeling and monitoring the active sites of ATPases and kinases. In its first application, the reagent is used to explore the active site of adenylate kinase from rabbit muscle. In the dark, AD-ATP inhibits adenylate kinase reversibly and competitively with KI = 0.25 +/- 0.01 microM. Under weak UV illumination, AD-ATP labels adenylate kinase irreversibly. The photoinactivation data also show KI = 0.25 +/- 0.02 microM. The ratio (r) of the specific activity of AD-ATP-labeled adenylate kinase to that of the unlabeled enzyme has been determined as a function of the number (n) of label/enzyme. The linear plot of r versus n with slope equal to -1 shows that the labeling is very specific, i.e. each label completely inactivates an enzyme molecule. After the labeled enzyme was partially hydrolyzed and the radioactive peptides analyzed and sequenced, it was found that Leu-115, Cys-25, and probably His-36 were labeled, in agreement with previous conclusions on the structure of the active site of this enzyme based on amino acid sequence, x-ray diffraction, and NMR studies. The environment-sensitive fluorescent dansyl group of AD-ATP can function as an in situ probe for monitoring ligand or conformation changes at the active site. The fluorescence of AD-ATP-labeled enzyme with n = 0.9 is not affected by ATP but increases with the concentration of AMP in solution. This observation is also in agreement with the previous conclusion that ATP does not bind to the AMP site of adenylate kinase. The observed enhancement of fluorescence indicates that binding of AMP by this enzyme causes environmental change at its ATP site. The possible usefulness of AD-ATP as an effective biological inhibitor or as a molecular probe for studying the structure and regulation of ATP-binding proteins is discussed.

Adenosine Triphosphate↗
PubMed1989

On the sensitivity of intact cells to perturbation by ethanol.

A comparison was made of ethanol's effects on the order of plasma membranes in intact cells and some isolated membrane preparations. Order was assessed by steady-state fluorescence polarization techniques using the non-permeant probe, TMA-DPH. The data show that two cultured cells, rat neonatal astroglial and N2A neuroblastoma, were sensitive to significant ethanol-induced disordering within the anesthetically relevant range (100 - 200 mM). Human erythrocytes, cultured fibroblasts and homogenized astroglial cells required higher ethanol concentrations (greater than 250 mM) to produce a similar effect. Intact erythrocytes were approximately twice as sensitive as erythrocyte ghost membranes to ethanol-induced perturbation. The neonatal glial and N2A cells were approximately five times more sensitive than synaptic membranes to ethanol effects. DMPC and DMPC + cholesterol liposomes and myelin membranes were insensitive to ethanol's effects. The incorporation of 10 mole % ganglioside GM1 sensitized the liposomes to ethanol-induced perturbation.

Cell Membrane↗
PubMed1989

The basis for intrinsic drug resistance or sensitivity to methotrexate.

Natural resistance to MTX in three different types of malignant cells is discussed. Two causes of resistance found were impaired drug uptake and impaired polyglutamylation. These resistance mechanisms have also been described to occur in cell lines with acquired resistance to this drug. In cells with acquired drug resistance, these phenotypic changes may reflect mutations in genes associated with transport of MTX or the enzyme DHFR, or in some circumstances reversion of the cell to a different phenotype without a mutational event. In cells with intrinsic resistance, the genetic mechanism responsible for these phenotypes is not known. Alternate treatment programs, e.g., with TMTX, or with TMTX and CPG2, a folate depleting enzyme, are under evaluation for these MTX resistant neoplasms.

Carcinoma, Squamous Cell↗
PubMed1989

Changes in desmosine and pyridinoline crosslinks during rapid synthesis and degradation of elastin and collagen in the rat uterus.

The wet weight of the rat uterus increased 8-fold during pregnancy and fell by 70% within 5 days postpartum. Uterine collagen increased about 5-fold during pregnancy and also fell by 70% within 5 days. The crosslink pyridinoline remained constant at 0.28 mole/mole collagen at every time point, with the possible exception of 11-12 days of pregnancy. The pyridinoline link can therefore form within the short time span of a few days, a feature presumed to be necessary to maintain the full mechanical strength of the uterus during labor. Uterine elastin increased about 8-fold during pregnancy, but the desmosines did not keep pace and fell from a normal value of 1.43 mole/mole elastin to a low of 0.89 at term. Moreover, elastin content reached a maximum several days prior to parturition and then declined continuously to 5 days postpartum. During this decline there was a selective loss of the poorly crosslinked elastin. The desmosines cannot be used as a direct measure of uterine elastin content, because of their continuously changing levels. Desmosines and pyridinoline were measured both by ELISA and by the amino acid analyzer. The two methods gave almost identical results when elastin and collagen were first separated from each other.

Amino Acids↗
PubMed1989

Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.

Animals↗
PubMed1989

Distinct adenosine 3',5'-monophosphate and phorbol ester-responsive signal transduction pathways converge at the level of transcriptional activation by the interactions of DNA-binding proteins.

Evidence is presented that distinct cellular signal transduction pathways involving cAMP-dependent protein kinase-A and phorbol ester-stimulated protein kinase-C coordinately modulate gene transcription through common as well as distinct cis-acting elements and DNA-binding proteins. When transfected and expressed in HeLa and placental JEG-3 cells, fusion reporter plasmids that differ only by a single base deletion or addition to interconvert the octameric cAMP-responsive element TGACGTCA (CRE) to form the heptameric phorbol ester-responsive element TGACTCA (TRE) are differentially regulated by cAMP and phorbol esters [12-O-tetradecanoyl phorbol-14-acetate (TPA)]. Transcription directed by the CRE is stimulated by cAMP and not TPA, although the basal expression mediated by this element in JEG-3 and HeLa cells is augmented by endogenous protein kinase-C activity. In contrast, TRE mediates transcriptional responses to both cAMP and TPA, and the two agents together give synergistic responses. Inhibition of cAMP-dependent protein kinase-A by expression of a minigene encoding a peptide inhibitor of A-kinase abolishes the response of TRE to cAMP alone as well as the cAMP-induced component of the synergistic response to treatment with both TPA and cAMP. Desensitization of the protein kinase-C dependent pathway by prolonged exposure of cells to phorbol esters eliminates the TPA-induced transcription by TRE and inhibits the TPA-induced component of the synergistic response to both cAMP and TPA. Therefore, both protein kinases, A and C, are involved in transcriptional activation by the TRE; the function of either kinase alone results in a moderate level of activity, but the combined results of both functionally stimulated kinases are synergistically positive. Electrophoretic mobility shift assays using whole extracts of JEG-3 cells indicate that a common factor(s) binds both TRE and CRE; however, another factor(s) that binds to the CRE will not bind to the TRE. Further, a latent regulatory enhancer element (URE) located upstream of the CRE's in the human alpha gonadotropin gene, although inactive when paired alone with the alpha 100 promoter, induces basal and stimulated transcriptional activity of both CRE and TRE on the average of 10- to 20-fold. The data support the existence of a gene regulatory network consisting of related cis-acting elements and DNA-binding proteins whose transcriptional activities are regulated by the convergent actions of protein kinases-C and -A.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

The MVV/FEV1 ratio in normal and asthmatic subjects.

We compared the ratio of maximal voluntary ventilation/forced expiratory volume in one second (MVV/FEV1) in normal individuals and asthmatic subjects. Our hypothesis was that MVV/FEV1 would be lower in asthmatic subjects. We also compared MVV/FEV1 results with the effects of deep inspiration (DI) on bronchomotor tone and with methacholine responsiveness. In normal subjects, MVV/FEV1 was higher than that in asthmatic subjects. Specific conductance (SGaw) increased after a single DI or an MVV maneuver in normal individuals. An MVV maneuver caused SGaw to decrease in asthmatic subjects. In asthmatic subjects, there was a significant correlation between MVV/FEV1 ratio and magnitude of decrease in SGaw after an MVV maneuver. Results of methacholine testing indicated that asthmatic subjects with low MVV/FEV1 ratios were more responsive to methacholine than those whose MVV/FEV1 ratios were within normal range. We conclude that MVV/FEV1 ratios are abnormally low in many asthmatic subjects and that low MVV/FEV1 ratios may indicate increased bronchial responsiveness to methacholine.

Adult↗
PubMed1989

[Solubilization and characterization of benzodiazepine receptors in frontal cortex of rabbit brain].

[3H]Flunitrazepam binding to benzodiazepine receptors solubilized by the detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate (CHAPS) was saturable and showed non-linear Scatchard plot with KD1 0.31 nmol/L and KD2 6.7 nmol/L. The affinities of soluble receptors to benzodiazepine were consistent with P2 membrane. One radioactive zone was found by SDS-PAGE after photoaffinity labelling of soluble membrane and the apparent molecular weight of 55,000 was reported. [3H]Flunitrazepam binding to soluble receptors was enhanced by GABA, NaCl or KCl and barbiturates, but inhibited by bicuculline and picrotoxinin. The enhancement of GABA on [3H]flunitrazepam binding was amplified by NaCl or KCl and antagonized by bicuculline and picrotoxinin. These results suggest that the benzodiazepine receptors solubilized by CHAPS have their pharmacological properties and are still associated with GABA receptors and chloride channel.

Animals↗
PubMed1989

Metabolism and disposition of (R)-4-[3-(2-hydroxy-2-phenyl)ethylamino-3-methylbutyl]benzamide Hcl (LY 195448) in rodents.

The disposition and metabolism of (R)-4-[3-(2-hydroxy-2-phenyl)ethylamino-3-methylbutyl]benzamide HCl, or LY 195448 (LY), were studied in rodents 1 hr following a single iv injection of 14C-LY at 30 mg/m2 (10 mg/kg mice and 5 mg/kg rats), In all tissues and carcases, recovery of 14C was nearly complete. The majority of radioactivity was recovered from intestine (40%) whereas liver and urine accounted for 10% each, kidney for 3-5%, and plasma for 1-3% of total administered radioactivity. Analyzing by HPLC and TLC, para-hydroxy-LY (para-OH-LY) in addition to unchanged drug, was found in all tissues. Meta-OH-LY was detected only in urine, intestine and kidney. These metabolites were found to be conjugated with either glucuronide or sulfate moieties. Peaks coeluting with authentic para-OH-meta-methoxy-LY and di-OH-LY were also observed.

Animals↗
PubMed1988

Dermal penetration of avermectin B1a in the rhesus monkey.

Forearms of rhesus monkeys were treated with [3H]avermectin B1a in three different vehicles and concentrations so that the penetration of avermectin B1a through skin could be determined. In order to simulate exposure of farm workers, such as mixer-loaders, applicators, and harvesters, to this pesticide, avermectin B1a was applied to the forearms of the monkeys as an emulsifiable concentrate (300 micrograms/monkey), a diluted emulsifiable concentrate (4.5 micrograms/monkey), and as a suspension in water (216 micrograms/monkey). After 1 or 10 hr of exposure, the treatment area was washed. The levels of radioactivity were determined in the urine, feces, plasma, and wash. On the basis of the amounts of radioactivity excreted in the urine and feces and the levels of radioactivity in the plasma after dermal application compared to those found after intravenous administration of the compound, less than 1% of the doses was absorbed. These data indicate that avermectin B1a would not readily penetrate the skin of farm workers exposed to it. Therefore, the hazard to farm workers exposed to this compound would be substantially mitigated.

Animals↗
PubMed1988

The expression of viral and cellular genes in papillomas of the choroid plexus induced in transgenic mice.

A line of transgenic mice that carry the SV40 gene for the large Tumor antigen express this protein during the first two weeks of life in brain tissue. By 30-40 days after birth, independently derived multiple foci of abnormal cells appear throughout the choroid plexus. After 90 days, higher levels of T antigen and rapid tumor growth are detected and all these animals die in a narrow time span, between 100-120 days. In situ hybridization with tissue sections and Northern blot analysis have been employed to follow the steady state levels of SV40 RNA and the p53 oncogene RNA levels in normal and tumor tissues. The level of SV40 RNA is quite variable between tumor cells in a section. This heterogeneity of T antigen mRNA levels could permit the selection of cells (from the multiple foci) expressing higher levels of T antigen and growing more rapidly. The increased levels of p53 RNA observed in tumor cells could then result from the active growth state of these cells or a more direct transcriptional activation. Two cellular genes, transthyretin and the 5-HT1C serotonin receptor, both of which are preferentially expressed in normal choroid plexus cells, were also examined for RNA production in these tumors of the choroid plexus. Both of these genes produced high levels of RNA in tumor tissue indicating the retention of well differentiated gene expression in these tumor tissues. This reflects, at the level of gene expression, the well differentiated morphology of these papillomas of the choroid plexus. Interestingly, as cell lines have been derived from these tumors, both the choroid plexus specific RNA species (for 5-HT1C receptor) and characteristic morphology were lost and an increase in T antigen levels was observed.

Animals↗
PubMed1988

Ultrastructural study of internalization and recycling of antigen by antigen presenting cells.

The evidence which suggests that a helper T (Th) cell recognizes a processed form of a soluble protein antigen in association with a class II MHC antigen on the surface of an antigen presenting cell (APC) has raised many questions and much controversy. A major question that remains unanswered is what is the cellular site(s) and mechanism(s) in which such an antigen is handled by an APC. The controversy relates to the issue of whether a protein antigen is required to be processed by an APC before it is presented to a Th cell. A currently favored hypothesis of antigen presentation, which stems mainly from analyses of T cell reactivity to peptides of protein antigens, is that such antigens are internalized by an APC, processed intracellularly, recycled to the cell surface and then presented to Th cells. As a first test of this hypothesis, we reasoned that although it is currently difficult to study the biochemistry of antigen processing, it is possible to study whether a protein antigen is internalized by an APC and recycled to its surface. In this report, colloidal gold conjugates of pork insulin (PI-Au), a tryptic peptide of pork insulin lacking the insulin receptor-binding portion of the molecule (TI-Au), myoglobin (MYO-Au), and apomyoglobin (APO-Au) were used to follow the pathway(s) and kinetics of antigen internalization and recycling in TA3 B hybridoma cells. Transmission electron micrographs of the routes followed by the PI-Au and TI-Au conjugates suggest that these antigens are internalized and recycled to the surface of an antigen presenting cell within 2-4 hr. In contrast, the patterns obtained for MYO-Au and APO-Au suggest that either the internalization of these antigens serves to channel them into a degradative pathway or that the kinetics of recycling of these antigens is slower. The two types of patterns observed may not be mutually exclusive and the purpose of internalization may vary, depending on the nature of the antigen. These data represent the first analysis of the kinetics and pathways of internalization and recycling of an antigen by an APC. It is impossible to formally prove that the pathway we have demonstrated is the one responsible for processing for antigen presentation. Nevertheless, these results support the notion that a protein antigen is handled intracellularly by an APC and recycled to the surface before it is presented to a Th cell.

Animals↗
PubMed1987

Viscoelastic models for enzymes with multiple conformational states.

In this article we represent an enzyme capable of exhibiting more than one conformational state as a viscoelastic unit embedded in a fluid medium. We show how this viscoelastic unit is thermally activated to make transitions between equilibrium states, and propose this model as a mesoscopic representation for transitions between conformational states of an enzyme. In this representation, kinetic constants for transitions between conformations are given explicitly in terms of interactions between the enzyme and the medium. Two applications of this model are discussed: mnemonic enzymes, and co-operative enzymes exhibiting half-of-the-sites reactivity.

Allosteric Regulation↗
PubMed1987

Rat cortical morphology following crowded-enriched living conditions.

This experiment studied cerebral cortical morphology in rats living in a crowded-enriched condition. Three groups of 60-day-old, male Long-Evans rats were divided accordingly: 12 rats, 3 per small cage (32 X 20 X 20 cm), standard colony condition; 12 rats in a single, large, enrichment cage with "toys" (70 X 70 X 45 cm), enriched condition; and 36 rats in a large, single, enrichment cage with "toys", crowded-enriched condition. Matched toys for the two enriched cages were changed twice a week at the time of cage cleaning. Measurements on 20-micron, transverse brain sections showed that in both the crowded-enriched and enriched groups the thickness of the medial occipital cortex increased by 4 to 6% compared with the cortex from animals in the standard colony condition. In addition, the crowded-enriched group demonstrated a 4% (P less than 0.05) increase in thickness in area 39 in the left hemisphere compared with the standard control. However, the thickness in area 39 in the crowded group was not significantly different from that of the enriched area 39. These results indicate that the cortex increases in thickness as much with "crowding" and enrichment as with enrichment alone. We hypothesize that diversion through interaction with "toys" mitigates the stress of crowded conditions.

Animals↗
PubMed1987

Transcholecystic endoscopic choledocholithotripsy: successful management of retained common bile duct stone.

Removal of large common bile duct stones has been a continuing challenge. The feasibility and efficacy of transcholecystic endoscopic choledocholithotripsy in a high-risk patient are demonstrated in this report. The procedure requires an established cholecystostomy track, catheter dilatation of the cystic duct, and the application of electrohydraulic shock waves to the calculus. The use of a choledochofiberscope permits the passage of the electrohydraulic probe and minimizes complications by direct monitoring. Stone fragments are removed by basket retrieval. This procedure in conjunction with minicholecystostomy may obviate the need for surgery in selected high-risk patients with combined gallbladder and common bile duct stones.

Cholelithiasis↗