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Biomedical subjects

J C Lee

Publications and source records attributed to J C Lee.

At least 19 recordsLinked to original sources
PubMed1992

Structure analysis of the 5' external transcribed spacer of the precursor ribosomal RNA from Saccharomyces cerevisiae.

Full-length precursor ribosomal RNA molecules were produced in vitro using as a template, a plasmid containing the yeast 35 S pre-rRNA gene under the control of the phage T3 promoter. The higher-order structure of the 5'-external transcribed spacer (5' ETS) sequence in the 35S pre-rRNA molecule was studied using dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, RNase T1 and RNase V1 as structure-sensitive probes. Modified residues were detected by primer extension. Data produced were used to evaluate several theoretical structure models predicted by minimum free-energy calculations. A model for the entire 5'ETS region is proposed that accommodates 82% of the residues experimentally shown to be in either base-paired or single-stranded structure in the correct configuration. The model contains a high degree of secondary structure with ten stable hairpins of varying lengths and stabilities. The hairpins are composed of the Watson-Crick A.T and G.C pairs plus the non-canonical G.U pairs. Based on a comparative analysis of the 5' ETS sequence from Saccharomyces cerevisiae and Schizosaccharomyces pombe, most of the base-paired regions in the proposed model appear to be phylogenetically supported. The two sites previously shown to be crosslinked to U3 snRNA as well as the previously proposed recognition site for processing and one of the early processing site (based on sequence homology to the vertebrate ETS cleavage site) are located in single-stranded regions in the model. The present folding model for the 5' ETS in the 35 S pre-rRNA molecule should be useful in the investigations of the structure, function and processing of pre-rRNA.

Base Sequence↗
PubMed1992

Energetics of allosteric regulation in muscle pyruvate kinase.

The regulatory mechanism of rabbit muscle pyruvate kinase has been studied as a function of temperature in conjunction with phenylalanine, the allosteric inhibitor. The inhibitory effect of phenylalanine is modulated by temperature. At low temperatures, the presence of phenylalanine is almost inconsequential, but as the temperature increases so does the phenylalanine-dependent inhibition of the kinetic activity. In addition, the presence of phenylalanine induces cooperativity in the relation between velocity and substrate concentration. This effect is especially pronounced at elevated temperature. The kinetic data were analyzed using an equation that describes the steady-state kinetic velocity data as a function of five equilibrium constants and two rate constants. Van't Hoff analysis of the temperature dependence of the equilibrium constants determined by nonlinear curve fitting revealed that the interaction of pyruvate kinase with its substrate, phosphoenolpyruvate, is an enthalpy-driven process. This is consistent with an interaction that involves electrostatic forces, and indeed, phosphoenolpyruvate is a negatively charged substrate. In contrast, the interaction of pyruvate kinase with phenylalanine is strongly entropy driven. These results imply that the binding of phenylalanine involves hydrophobic interaction and are consistent with the basic concepts of strengthening of the hydrophobic effect with an increase in temperature. The effect of phenylalanine at high temperatures is the net consequence of weakening of substrate-enzyme interaction and significant strengthening of inhibitor binding to the inactive state of pyruvate kinase. The effects of salts were also studies. The results show that salts also exert a differential effect on the binding of substrate and inhibitor to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1992

Solution studies on the structure of bent DNA in the cAMP receptor protein-lac DNA complex.

Cyclic AMP receptor protein is involved in the regulation of more than 20 genes. A step in the mechanism of activation of transcription is to induce a significant bending of the DNA upon complex formation between specific DNA and the protein. The induced DNA bending and a structure of the protein-DNA complex were studied by fluorescence energy transfer in 50 mM Tris, 1 mM EDTA, and 50 mM KCl at pH 7.8 and 20 degrees C. The symmetry of the DNA bend was estimated by measuring the efficiency of transfer between the protein and a label on either the upstream or the downstream end of a lac DNA fragment. The results show that the bend, despite the asymmetry in the DNA sequence, is symmetrical, for the fragments which length ranges from 26 to 40 bp. Using fluorescence energy transfer, the extent of DNA bending was estimated by measuring the end-to-end distance of the DNA fragment which was labeled with a donor-acceptor pair on two opposite ends. Both steady-state and time-resolved measurements showed that in a 26 bp lac DNA fragment complexed with cyclic AMP receptor protein, the end-to-end distance is about 77 A which corresponds to a bending angle of 80 degrees or 100 degrees, depending on the actual contour length between the fluorophores in the free DNA fragment. The results using longer DNA fragments show no measurable amount of energy transfer; thus, it is very unlikely that the DNA completely wraps around the CRP molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Intersubunit communications in Escherichia coli cyclic AMP receptor protein: studies of the ligand binding domain.

Escherichia coli cAMP receptor protein (CRP) is a homodimer in which each subunit is composed of two domains. The C-terminal domain is responsible for DNA recognition, whereas the larger N-terminal domain is involved in cAMP binding. Biochemical and genetic evidence suggests that both intersubunit and interdomain interactions play important roles in the regulatory mechanism of this protein. Essentially all intersubunit contacts occur via a long C-helix which is a part of the N-terminal domain. In this work, intersubunit interactions in CRP were studied with the use of two proteolytic fragments of the protein. Subtilisin digestion produces a fragment (S-CRP) which includes residues 1-117 and in which about 85% of the C-helix is removed, whereas chymotrypsin digestion produces a fragment (CH-CRP) consisting of residues 1-136, in which the whole C-helix is preserved. Both fragments were purified and subjected to functional tests which included cAMP binding, subunit assembly, and hydrodynamic properties in the presence and absence of cAMP. S-CRP binds cAMP with a similar affinity to that of native CRP but with reduced cooperativity. CH-CRP exhibits about 1 order of magnitude tighter binding of cAMP than S-CRP or CRP and the highest degree of negative cooperativity. Both fragments are dimeric with dimerization constants around 10(8) M-1. Ligand binding promotes dimerization and induces a small contraction of both S-CRP and CH-CRP. There is no apparent correlation between dimer stability and cooperativity of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Global conformational changes in allosteric proteins. A study of Escherichia coli cAMP receptor protein and muscle pyruvate kinase.

One of the basic features in allosteric regulation involves long range transduction of information. Based on crystallographic data on protein systems that are regulated by allosteric mechanisms, a global conformational change has always been observed. It is, therefore, important and useful to correlate the cooperativity of global structural change with the mode of binding of the regulatory ligand. Two systems were chosen for study, namely Escherichia coli cAMP receptor protein and muscle pyruvate kinase, which show negative and positive cooperativity in the binding of allosteric ligands, respectively. Quantitative titration of the global structural change, monitored by a high precision analytical gel chromatography technique, was conducted as a function of allosteric effector concentration. The results obtained for cAMP receptor protein show that the protein undergoes contraction upon binding of cAMP. The decreases in Stokes radius associated with complex formation are 0.1 +/- 0.1 and 0.7 +/- 0.1 A when one and two cAMP-binding sites are filled, respectively. The results for the pyruvate kinase system show a concerted structural change that quantitatively match the predicted behavior based on equilibrium constants derived from the analysis of steady state kinetic data by a two-state model. Hence, for these two systems, these results show that negative and positive cooperativity are correlated with sequential and concerted modes of structural change, respectively.

Allosteric Regulation↗
PubMed1992

Sulfhydryl groups on yeast ribosomal proteins L7 and L26 are significantly more reactive in the 80 S particles than in the 60 S subunits.

The sulfhydryl-directed fluorescent reagent, 5-iodoacetamidofluorescein (IAF), reacts differently with proteins from the 60 S ribosomal subunit of Saccharomyces cerevisiae when this subunit is free as opposed to being contained within the 80 S ribosome. When the 80 S ribosomes and the free 60 S subunits were labeled with IAF, the specific fluorescence intensity (fluorescence intensity unit/A260 60 S subunit) of the subsequently derived 60 S was 16.3 and 5.4, respectively. Gel analysis showed that proteins L7 and L26 were selectively labeled and contained greater than 90% of the total fluorescent label, when 80 S ribosomes were labeled. When free 60 S subunits were labeled, six additional proteins were labeled. Both types of modified 60 S subunits were equally capable to support protein synthesis in vitro. Reassociation of the IAF-labeled derived and free 60 S subunits with unmodified 40 S subunits resulted in a maximum of 5-7% decrease and a 3-fold increase, respectively, in the fluorescence intensity without a shift in the emission maxima. The data suggest that ribosomal proteins L7 and L26 contain SH groups that respond to ribosomal subunit association and become more reactive in the intact ribosome than in the subunit. The environments of some or all of the additionally labeled proteins are also sensitive to subunit reassociation.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1992

HLA-DRB3 gene alleles in Caucasian patients with Graves' disease.

Graves' disease (GD) is a human leukocyte antigen (HLA) linked organ-specific autoimmune disease. In German GD patients the disease is associated with HLA specificities of the HLA-DRw52 family (HLA-DR3, -DR5, and DR6; HLA-DRB3 positive HLA haplotypes). Recently, a strong association with a HLA-DRB3 restriction fragment length polymorphism gene has been described. To study HLA-DRB3 alleles and their association with the disease, a large cohort of controls (n = 3724) and GD patients (n = 304) was analyzed. HLA-DR allelic combinations revealed an increase in HLA-DR3/DR5 heterozygous patients (relative risk 2.9; P < 0.001). HLA-DRB3 alleles, as defined by DNA typing in HLA-DR matched groups revealed a significant increase in DRB3*0101 homozygosity (relative risk 17.5; P < 0.001) in HLA-DR3 homozygous patients. In GD patients with ophthalmopathy (grade II or higher, according to Werner) DRB3*0101/*0202 heterozygosity revealed an increased relative risk of 5.5 (P < 0.001). Non-HLA-DR3 homozygous, DRB3*0101/*0202 heterozygous patients were at the highest risk for endocrine ophthalmopathy (relative risk 10; P < 0.001). Our data, based on DNA typing methods of HLA-D genes, provide evidence that the susceptibility is strongly associated with HLA-DRB3 genes.

Alleles↗
PubMed1992

Yeast 5S rRNA binding to ribosomal protein L1a alters the fluorescence of tryptophan residues lying outside the binding site.

The yeast ribosomal protein L1a contains two tryptophan residues located at positions 95 and 183. Spectrofluorometric analysis showed that the average tryptophan environment is moderately polar. Quenching studies of the yeast 5S rRNA-L1a protein complex (RNP) with acrylamide and iodide revealed tryptophan heterogeneity. The two tryptophan residues are located in the non-RNA-binding region of the L1a molecule. However, dissociation of the yeast 5S rRNA-L1a protein RNP complex to its components resulted in a decline of tryptophan fluorescence. The observation implied that the environment of the tryptophan-containing L1a regions which were not known to be involved in RNA binding was influenced by association with the 5S rRNA molecule.

Acrylamide↗
PubMed1992

W-7, a calmodulin antagonist, and contracture of malignant hyperthermia susceptible skeletal muscle.

1. In malignant hyperthermia susceptible muscle fibers, the calmodulin antagonist, W-7 (10 microM), evoked contractures and potentiated halothene (3%) induced contracture. No effect was seen at 0.1 or 1.0 microM) W-7. 2. Dantrolene sodium (6 microM) prevented and reversed W-7 induced contracture: nifedipine did not. 3. In chemically skinned fibers, 10 microM, 1.0 microM, and 0.1 microM W-7 released 100%, 30%, and 10% of stored calcium respectively, and the effect of 10 microM W-7 was irreversible in that the SR was unable to re-sequestor calcium after exposure to the drug. 4. The release of calcium by W-7 was not prevented by exogenously added calmodulin (3 microM), nor mimicked by mastoparan (10 microM). 5. Calcium release by W-7 appears to be independent of calmodulin inhibition.

Animals↗
PubMed1992

Altered mechanical responses of malignant hyperthermic skeletal muscle during repetitive stimulation.

This investigation examined the mechanical responses of malignant hyperthermic (MH) and normal porcine skeletal muscle to repetitive stimulation. Twitch and maximal tetanic tensions were not significantly different between muscle types. Tensions produced during stimulation at 20-80 Hz were significantly less in MH muscle than in normal muscle. In addition, MH muscle showed significantly greater force decline (tetanic fade) at the end of contractions evoked by 20-80 Hz stimulation. When stimulated to fatigue, both normal and MH muscle exhibited similar rates of tension decline during the initial minutes. Further stimulation caused additional decline in normal muscle, but a tension plateau in MH muscle. In all cases, normal muscle had greater magnitudes of fatigue than did MH muscle. Results show that there are marked differences between MH and normal muscle in the mechanical responses to repetitive stimulation. Due to its inability to properly regulate intracellular Ca2+ exchange, it is possible that MH muscle might be a useful tool for identifying the mechanisms of muscle fatigue in normal muscle.

Animals↗
PubMed1992

Opioid antagonism, perceived exertion and tolerance to exercise-thermal stress.

In an attempt to investigate the physiological responses to opioid receptor blockade during exercise in the heat, five male volunteers completed two bouts of stationary cycling at 70% VO2max in a hot (33 degrees C765% RH) environment. Exercise was conducted following the administration of either naloxone or saline (4 mg i.v.) five minutes prior to exercise. A second 4 mg dose was administered at 25 minutes of exercise. Performance time was 11% shorter (p = 0.06), and RPE response was significantly higher at test termination on naloxone. No drug effect was observed on rectal or mean skin temperature during exercise. Forearm blood flow (FBF) was higher on naloxone, while exercise heart rates were lower on the drug versus saline. No significant changes were observed in estimated mean arterial pressure or gross sweat responses to exercise. Plasma immunoreactive beta-endorphin was significantly elevated in the naloxone trial only. Thus, while opioids may play some hemodynamic role during exercise in the heat, it appears that opioid mediation of the perceived stress of exercise contributes more to an individual's thermal tolerance. Additionally, the results suggest that perceptual and hemodynamic/cardiovascular responses that may be mediated by these peptides are dissociable phenomena.

Adult↗
PubMed1992

Effect of a human immunodeficiency virus protease inhibitor on human monocyte function.

Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). Encoded by the HIV genome are several precursor proteins that undergo proteolytic cleavage to yield functional proteins. The env precursor protein is cleaved by a cellular protease. The gag precursor protein of HIV (p55), however, is cleaved by a virally encoded aspartate protease (HIV Protease). Cleavage of p55 is required for viral maturation and infectivity. There are also several host cell aspartate proteases that serve important homeostatic functions. Cathepsins D and E are lysosomal aspartate proteases which are believed to play an important role in macrophage function, and it has been suggested that inhibition of these enzymes by an HIV protease inhibitor may exacerbate immunosuppression in AIDS patients. We have studied the effect of SK&F 107461 (a hydroxyethylene dipeptide isostere inhibitor of HIV protease), on various host defense functions of human monocytes. Pepstatin A (an inhibitor of most aspartate proteases) and leupeptin (an inhibitor of serine and cysteine proteases) were included as controls. Although less potent than the prototypic aspartate protease inhibitor pepstatin, SK&F 107461 inhibited partially purified cathepsin D in vitro. However, in cell-based assays, SK&F 107461 had no effect on the degradation of hemoglobin, antigen processing of the protein antigen streptokinase, or secretion of 17-kD IL-1 beta by monocytes at concentrations which inhibit maturation of intracellular virus in HIV infected monocytes. Furthermore, SK&F 107461 had no effect on constitutive candidacidal activity. In contrast, leupeptin and pepstatin A partially inhibited accessory cell function of monocytes in the proliferative response to the recall antigen streptokinase. In addition, leupeptin partially inhibited degradation of hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-Presenting Cells↗
PubMed1992

Staphylococcus aureus microcapsule expression attenuates bacterial virulence in a rat model of experimental endocarditis.

The virulence of the Staphylococcus aureus strains that differed only in capsule expression was compared in a rat model of catheter-induced experimental endocarditis. The ID50 of all the strains was low (less than 3 x 10(3) cfu of S. aureus), suggesting that this model may be more sensitive than other animal models to differences in bacterial virulence. Compared with the wild-type strains that expressed type 5 or type 8 capsular polysaccharides, mutant strains devoid of capsule had significantly lower ID50 values. In contrast, a mutant that produced scant amounts of the type 5 polysaccharide had an ID50 similar to that of the parental type 5 isolate. As the bacterial inoculum was increased, each of the S. aureus strains reached final concentrations of 10(10)-10(11) cfu/g of vegetation; however, the nonencapsulated mutants colonized the left-sided vegetations at lower inocula than did the wild-type strains. This study indicates that microcapsule expression attenuates bacterial virulence in a rat model of catheter-induced endocarditis.

Animals↗
PubMed1992

Platelet imipramine binding in depressed children and adolescents.

Kinetic constants of platelet imipramine binding were determined in youths with major depression, and a contrast group. Subjects actively depressed (N = 10) had significantly fewer imipramine binding sites (Bmax) (877 +/- 148 fmol/mg protein) than recovering depressives (N = 12) (1220 +/- 428 fmol/mg protein) and contrasts (N = 10) (1270 +/- 230 fmol/mg protein). Affinity constants (Kd) (1.14 +/- 0.36 nM, 0.97 +/- 0.31 nM, and 1.17 +/- 0.39 nM, respectively) were similar among the groups. Actively depressed males but not females had fewer imipramine binding sites than both their sex-matched comparison groups. Although actively depressed females' Bmax was significantly lower than recovering depressed and nondepressed males, neither age, Tannner stage, nor circannual rhythms influenced Bmax, but suicidality may be associated with low Bmax. A decrease in Bmax may be a state-specific marker of major depression in boys or associated with a depressive disorder with a suicidal history.

Adolescent↗
PubMed1992

Phagocytic killing of encapsulated and microencapsulated Staphylococcus aureus by human polymorphonuclear leukocytes.

Phagocytosis by human polymorphonuclear leukocytes (PMNs) is an important host defense against infections caused by Staphylococcus aureus. Using an in vitro assay, we compared the opsonic requirements for phagocytic killing of prototype strains of encapsulated (type 1) and microencapsulated (type 5 and type 8) S. aureus by human PMNs. More than 85% of broth-grown, logarithmic-phase type 5 and 8 S. aureus organisms were killed by PMNs incubated with fresh normal human, rabbit, or guinea pig serum with complement activity. Under similar conditions, the highly encapsulated type 1 strain was not killed. Both encapsulated and microencapsulated strains were opsonized for phagocytosis by heat-inactivated serum raised in rabbits to killed bacteria. Opsonization by homologous serum was required for phagocytosis of the type 1 strain. In contrast, microencapsulated type 5 and 8 S. aureus organisms were killed by heat-inactivated rabbit serum raised to type 5, type 8, or nonencapsulated isolates; this result suggested that antibodies to the capsule or to cell wall components other than the capsule could opsonize these organisms for phagocytosis. The specificity of the assay was confirmed with capsule type 5-specific monoclonal antibodies, which were opsonic only for the type 5 S. aureus isolate. These studies indicate that, unlike the highly encapsulated type 1 strain, broth-grown microencapsulated S. aureus strains do not resist opsonophagocytic killing in vitro by normal serum.

Colony-Forming Units Assay↗
PubMed1992

Glomerulonephritis associated with Robertsonian translocation t(13;14).

We report the first case of glomerulonephritis associated with Robertsonian translocation t(13;14) in a 18-year-old female. The renal biopsy revealed mesangial proliferative glomerulonephritis with mesangial IgG and C3 deposits. The patient also has congenital absence of ovaries and an underdeveloped uterus. Our report suggests that renal abnormalities may be found in patients with Robertsonian translocation.

Adolescent↗
PubMed1992

PCR amplified HLA DQ alpha and DQ beta DNA typed by dot-blot hybridization and direct sequence for suspect.

For more effective screening of suspects, we combined two methods to individualize the biologic evidence from a crime scene: polymerase chain reaction (PCR) dot-blot hybridization for DNA typing of HLA Dq alpha; and PCR direct sequencing for DNA typing of HLA DQ beta. PCR-DQ alpha was typed by an AmpliType HLA DQ alpha kit from Cetus. PCR-DQ beta was typed by direct sequence using the dideoxy chain termination method. In 20 DNA samples, complete separation was demonstrated by these two polymorphic DNAs. This will be of considerable use in screening suspects in crime investigation work. The sensitivity of PCR is useful for trace evidence, and the rapid results of dot-blot hybridization and the precise results of direct sequencing have significant advantages.

Base Sequence↗