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Biomedical subjects

K Chu

Publications and source records attributed to K Chu.

At least 73 records · Page 4Linked to original sources
PubMed1994

Requirement for kinase activity of CD4-associated p56lck in antibody-triggered T cell signal transduction.

The lymphoid-specific Src family protein tyrosine kinase p56lck (Lck) is non-covalently associated with the cytoplasmic tail of CD4 and has an essential role in T cell activation. Engagement of ligand by the T cell antigen receptor (TCR) is followed by rapid tyrosine phosphorylation of several cellular proteins, including phospholipase C gamma 1 (PLC) and the TCR-associated CD3 zeta polypeptides. Tyrosine phosphorylation of PLC gamma 1 results in activation of PLC and subsequent phosphatidylinositol turnover. We have studied the effects of the CD4-associated Lck molecule on TCR-mediated activation of the protein tyrosine kinase (PTK) pathway in a murine T cell hybridoma. Antibodies against CD3 elicited the expected PTK activation, which was enhanced upon co-cross-linking of CD4. In contrast, anti-TCR-alpha beta antibodies had no effect on the PTK pathway unless CD4 was co-cross-linked. Antibody cross-linking of CD4 alone failed to induce the same pattern of tyrosine phosphorylation. Similar results were obtained when a chimeric protein consisting of the extracellular and transmembrane domains of CD4 linked to the intracellular Lck molecule was used in place of CD4. The tyrosine kinase activity of Lck was essential for the activity of the chimeric protein. Cross-linking of the CD4/Lck chimera to a CD8/zeta chimeric molecule also facilitated induction of the PTK pathway with anti-CD8 antibodies. Moreover, the interaction of the two chimeric proteins, either in vitro or in vivo, resulted in tyrosine phosphorylation of CD8/zeta. The effects of CD4/Lck on tyrosine phosphorylation and activation of PLC correlated well with the effects on PTK activation. Our results suggest that the Lck molecule positively regulates the TCR-coupled PTK pathway by phosphorylating tyrosines on the TCR-associated CD3 zeta polypeptides.

Animals↗
PubMed1994

Severe factor VII deficiency caused by mutations abolishing the cleavage site for activation and altering binding to tissue factor.

Factor VII (F.VII) is a vitamin-K-dependent serine protease required in the early stages of blood coagulation. We describe here a patient with severe F.VII deficiency, with a normal plasma F.VII antigen level (452 ng/mL) and F.VII activity less than 1%, who is homozygous for two defects: a G-->A transition at nucleotide 6055 in exon 4, which results in an Arg-->Gln change at amino acid 79 (R79Q); and a G-->A transition at nucleotide 8961 in exon 6, which results in an Arg-->Gln substitution at amino acid 152 (R152Q). The R79Q mutation occurs in the first epidermal growth factor (EGF)-like domain, which has previously been implicated in binding to tissue factor. The R152Q mutation occurs at a site (Arg 152-Ile 153) that is normally cleaved to generate activated F.VII (F.VIIa). Analysis of purified F.VII from patient plasma shows that the material cannot be activated by F.Xa and cofactors. In addition, in an in vitro binding assay using relipidated recombinant tissue factor, patient plasma showed markedly reduced binding to tissue factor at all concentrations tested. In an effort to separate the contributions of the two mutations, three recombinant variants, wild-type, R79Q, and R152Q, were prepared and analyzed. The R152Q variant had markedly reduced activity in a clotting assay, whereas R79Q showed a milder, concentration-dependent reduction. The R152Q variant exhibited nearly normal binding in the tissue factor binding assay, whereas the R79Q variant had markedly reduced binding. The time course of activation of the R79Q variant was slowed compared with wild-type. Our results suggest that the first EGF-like domain is required for binding to tissue factor and that the F.VII zymogen lacks activity and requires activation for expression of biologic activity.

Adult↗
PubMed1994

Quadrupole gradient coil design and optimization: a printed circuit board approach.

Three different dual-axis quadrupole gradient coils for quantitative high resolution MR imaging of small animals, phantoms and specimens were designed and built using printed circuit board technology. Numerical optimization of the conductor positions was used to increase the volume of 0.4% gradient uniformity by up to a factor of four. In one coil, the volume of 5% gradient uniformity occupied 88% and 83% of the overall diameter and length of the coil, respectively. A systematic error of 0.5% in the wire placement was shown to cause a reduction in the volume of 0.4% gradient uniformity by a factor of two, though the region of 5% gradient uniformity was not significantly affected. Heat transfer calculations were used to determine maximum peak and root-mean-squared currents that could safely be applied to the coils.

Copper↗
PubMed1993

CD4 function in thymocyte differentiation and T cell activation.

The ectodomains of the T cell surface glycoproteins CD4 and CD8 bind to membrane-proximal domains of MHC class II and class I molecules, respectively, while both cytoplasmic domains interact with the protein tyrosine kinase (PTK) p56lck (lck) through a shared cysteine-containing motif. Function of CD4 and CD8 requires their binding to the same MHC molecule as that recognized by the T cell antigen receptor (TCR). In vitro studies indicate that CD4-associated lck functions even in the absence of kinase activity. In vivo experiments show that, whereas helper T cell development is impaired in CD4-deficient mice, high level expression of a transgenic CD4 that cannot bind lck rescues development of this T cell subset. These studies suggest that CD4 is an adhesion molecule whose localization is regulated through protein-protein interactions of the associated PTK and whose function is to increase the stability of the TCR signalling complex by binding to the relevant MHC. The function of CD4 in development has been further studied in the context of how double positive (CD4+CD8+) thymocytes mature into either CD4+ T cells with helper function and TCR specificity for class II or into CD8+ T cells with cytotoxic function and specificity for class I. Studies using CD4-transgenic mice indicate that development of single positive T cells involves stochastic downregulation of either CD4 or CD8, coupled to activation of a cytotoxic or helper program, respectively, and subsequent selection based on the ability of the TCR and remaining co-receptor to engage the same MHC molecule.

Animals↗
PubMed1993

Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin.

Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substrates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate.

Animals↗
PubMed1993

Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin.

Fouier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substrate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm. The resulting linear dichroism in the CO stretch spectrum yielded angles alpha > 20 degrees between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the alpha of the CO ligand.

Amino Acid Sequence↗
PubMed1992

Infrared study of the L, M, and N intermediates of bacteriorhodopsin using the photoreaction of M.

Infrared spectroscopy is used to characterize the transitions in the photocycle of bR involving the M intermediate. It has been shown previously that in this part of the photocycle a large protein conformational change takes place that is important for proton pumping. In this work we separate the spectra of the L, M, and N intermediates in order to better describe the timing of the molecular changes. We use the photoreaction of the M intermediate to separate its spectrum from those of L and N. At temperatures between 220 and 270 K a mixture of M and L or N is produced by illumination with green light. Subsequent blue illumination selectively drives M back into the ground state and the difference between the spectra before and after blue excitation yields the spectrum of M. Below about 250 K and L/M mixture is separated; at higher temperatures an M/N mixture is seen. We find that the spectrum of M is identical in the two temperature regions. The large protein conformational change is seen to occur during the M to N transition. Our results confirm that Asp-96 is transiently deprotonated in the L state. The only aspartic protonation changes between M and bR are the protonation of Asp-85 and Asp-212 that occur simultaneously during the L to M transition. Blue-light excitation of M results in deprotonation of both. The results suggest a quadrupolelike interaction of the Schiff base, Asp-85, Asp-212, and an additional positive charge in bR.

Bacteriorhodopsins↗
PubMed1992

Halogenated analogues of tamoxifen: synthesis, receptor assay, and inhibition of MCF7 cells.

This study was conducted to develop a ligand for imaging estrogen-receptor-positive breast tumors by positron emission tomography or single photon emission computed tomography. We synthesized fluoro and iodo analogues of tamoxifen, and these halogenated analogues produced greater affinity for binding to the receptor than tamoxifen. Values of the inhibition affinity constants were as follows: tamoxifen, 15,000 nM; fluoromethyl-N,N-diethyltamoxifen, 2500 nM for the cis isomer and 500 nM for the trans isomer; and iodomethyl-N,N-diethyltamoxifen, 1500 nM for the cis isomer and 1000 nM for the trans isomer. In studies of human MCF7 breast tumor cell growth, concentrations that inhibited tumor growth in 50% of the cases were as follows: tamoxifen, 11 microM; fluoromethyl-N,N-diethyltamoxifen, 4.5 and 11.8 microM for the cis and trans isomers, respectively; and iodomethyl-N,N-diethyltamoxifen, 2.4 and 6.3 microM for the cis and trans isomers, respectively. These studies suggest that both fluoro and iodo analogues of tamoxifen may be useful diagnostic compounds for predicting the response of estrogen-receptor-positive breast tumors to tamoxifen analogues used in chemotherapy.

Animals↗
PubMed1992

Determination of rate distributions from kinetic experiments.

Rate processes in proteins are often not adequately described by simple exponential kinetics. Instead of modeling the kinetics in the time domain, it can be advantageous to perform a numerical inversion leading to a rate distribution function f(lambda). The features observed in f(lambda) (number, positions, and shapes of peaks) can then be interpreted. We discuss different numerical techniques for obtaining rate distribution functions, with special emphasis on the maximum entropy method. Examples are given for the application of these techniques to flash photolysis data of heme proteins.

Carbon Monoxide↗
PubMed1991

Ligand binding to heme proteins: connection between dynamics and function.

Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (approximately 13,000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb*----Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb*----Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta chains of human hemoglobin and hemoglobin Zürich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme.

Binding Sites↗
PubMed1991

Developmental changes in vascular responses to histamine in normoxic and hypoxic lamb lungs.

This study of newborn (3-10 day old) and juvenile (6-8 mo old) in situ isolated lamb lungs was undertaken to determine whether 1) histamine receptor blockade accentuates hypoxic pulmonary vasoconstriction more in newborns than in juveniles, 2) histamine infusion causes a decrease in both normoxic pulmonary vascular resistance and hypoxic pulmonary vasoconstriction in newborns, and 3) the H1-mediated dilator response to infused histamine in newborns is due to enhanced dilator prostaglandin release. Pulmonary arterial pressure (Ppa) was determined at baseline and in response to histamine (infusion rates of 0.1-10.0 micrograms.kg-1 min-1) in control, H1-blocked, H2-blocked, combined H1- and H2-blocked, and cyclooxygenase-inhibited H2-blocked lungs under "normoxic" (inspired O2 fraction 0.28) and hypoxic (inspired O2 fraction 0.04) conditions. In newborns, H1-receptor blockade markedly accentuated baseline hypoxic Ppa, and H2-receptor blockade caused an increase in baseline normoxic Ppa. In juveniles, neither H1 nor H2 blockade altered baseline normoxic or hypoxic Ppa. Histamine infusion caused both H1- and H2-mediated decreases in Ppa in normoxic and hypoxic newborn lungs. In juvenile lungs, histamine infusion also caused H2-mediated decreases in Ppa during both normoxia and hypoxia. During normoxia, histamine infusion caused an H1-mediated increase in normoxic Ppa in juveniles as previously seen in mature animals; however, during hypoxia there was an H1-mediated decrease in Ppa at low doses of histamine followed by an increase in Ppa. Combined histamine-receptor blockade markedly reduced both dilator and pressor responses to histamine infusion. Indomethacin failed to alter the H1-mediated dilator response to histamine in newborns.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Development of the pulmonary vasculature in newborn lambs: structure-function relationships.

Our objectives were 1) to describe the quantitative light microscopy and ultrastructure of newborn lamb lungs and 2) to correlate hemodynamic changes during normoxia and hypoxia with the morphology. By light microscopy, we measured the percent muscle thickness (%MT) and peripheral muscularization of pulmonary arteries and veins from 25 lambs aged less than 24 h, 2-4 days, 2 wk, and 1 mo. At the same ages, lungs were isolated and perfused in situ and, after cyclooxygenase blockade with indomethacin, total, arterial (delta Pa), middle (delta Pm), and venous pressure gradients at inspired O2 fractions of 0.28 (mild hyperoxia) and 0.04 (hypoxia) were determined with inflow-outflow occlusion. During mild hyperoxia, delta Pa and delta Pm fell significantly between 2-4 days and 2 wk, whereas during hypoxia, only delta Pm fell. The %MT of all arteries (less than 50 to greater than 1,000 microns diam) decreased, and peripheral muscularization of less than 100-microns-diam arteries fell between less than 4 days and greater than 2 wk. Our data suggest that 1) the %MT of arteries determines normoxic pulmonary vascular resistance, because only arterial and middle segment resistance fell, 2) peripheral muscularization is a major determinant of hypoxic pulmonary vasoconstriction, because we observed a fall with age in peripheral muscularization of less than 100-micron-diam arteries and in delta Pm with hypoxia, and 3) the arterial limit of the middle segment defined by inflow-outflow occlusion lies in 100- to 1,000-microns-diam arteries.

Age Factors↗
PubMed1991

Developmental changes in effects of histamine on segmental pulmonary vascular resistances.

In mature animals histamine infusion typically causes an H1-mediated increase and H2-mediated decrease in pulmonary vascular resistance (PVR). Moreover, low histamine concentrations can cause H1-mediated relaxation of vascular strips in mature animals, and in newborn animals histamine infusion causes only H1-mediated decreases in PVR. The mechanisms responsible for the different H1-mediated responses are unknown. We used an inflow-outflow occlusion technique to identify the sites of H1- and H2-mediated responses in lungs of developing lambs. Histamine was infused at 1.0 and 10.0 micrograms.kg-1.min-1 in control and H1- and H2-blocked lungs of newborn and juvenile lambs under "normoxic" and hypoxic conditions and in hypoxic H2-blocked lungs of mature sheep. In newborns histamine caused significant H1-mediated decreases in resistance across the arterial (delta Pa) and middle (delta Pm) segments of the circuit during both normoxia and hypoxia. In normoxic juveniles low-dose histamine caused H1-mediated decreases in the resistance across delta Pa and delta Pm, but the resistances across delta Pm rose above baseline at the higher dose. The venous segment exhibited only a high-dose increase in resistance. During hypoxia, the high-dose H1-mediated pressor response of delta Pm was attenuated compared with that during normoxia; however, the increase in venous resistance was unaffected. In hypoxic mature sheep, no low dose H1-mediated decrease in segmental resistances was seen, but at the higher dose an increase in all resistances occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1989

Hepatitis B virus X gene activates kappa B-like enhancer sequences in the long terminal repeat of human immunodeficiency virus 1.

The role of the hepatitis B virus (HBV) X gene during virus infection has not been defined. We previously showed that expression of the HBV X gene in the human hepatocellular carcinoma cell line HepG2 trans-activates chloramphenicol acetyltransferase gene expression under control of the human immunodeficiency virus 1 (HIV-1) long terminal repeat and we have now identified a specific sequence in the HIV-1 long terminal repeat that is responsive to the HBV X gene. Plasmid constructs with the chloramphenicol acetyltransferase gene regulated by an isolated and twice-repeated 12-base-pair HIV-1 enhancer sequence homologous to the nucleotide sequence that binds the nuclear transcription factor NF-kappa B (the HIV-1 kappa B-like sequence) were trans-activated by the HBV X gene in HepG2 cells, indicating that the kappa B-like enhancer sequence in the HIV-1 long terminal repeat is responsive to the X gene. When eight copies of the HIV-1 kappa B-like sequence were used to regulate beta-globin gene expression, transcription of this gene was activated by the HBV X gene in HepG2 cells and no beta-globin gene transcription was detected in the absence of the HBV X gene. beta-globin gene expression regulated by the activator protein 2 (AP-2) binding sequence was not activated by the HBV X gene. Treatment of HepG2 cells with phorbol ester resulted in modest activation of the HIV-1 kappa B-like enhancer sequence suggesting that an NF-kappa B-like factor was induced in these cells as it is in T lymphocytes by phorbol ester; however, phorbol ester did not demonstrably enhance the activation of the HIV-1 enhancer observed with the HBV X gene. These experiments indicate that the HIV-1 kappa B-like transcriptional enhancer sequence is activated by the HBV X gene and suggest that the HBV X gene might play a role in regulating transcription of a gene under control of a kappa B-like enhancer during HBV infection. Since such a sequence has not been found in the HBV genome and HBV gene expression appears not to be regulated by the HBV X gene, a cellular gene that plays a role in HBV replication could be the target of the X gene during HBV infection.

Base Sequence↗
PubMed1985

An analysis of lung cancer risk from exposure to hexavalent chromium.

Lung cancer risk in relation to airborne levels of hexavalent chromium was analyzed for chromium chemical production workers studied by Hayes, Lilienfeld, and Snell. Hayes et al [18] had observed statistically significant increases in lung cancer mortality among chromium chemical production workers hired during 1945-1959 and followed to mid-1977. A dose-response was observed by Hayes et al. in that long-term workers had a higher lung cancer risk than short-term workers. Concurrent exposure data for the study plant were abstracted from the records of a local health department. The usual air concentration of hexavalent chromium was estimated as 413 micrograms per cubic meter (micrograms/m3) during 1945-1949. Cumulative exposure estimates for individual workers could not be developed with available information. Instead, cumulative exposures in terms of micrograms/m3-years were estimated for groups of short-term workers and long-term workers (cumulative exposure = usual exposure level in micrograms/m3 X average length of exposure). For workers hired during 1945-1949, cumulative exposures were estimated as 670 and 3,647 micrograms/m3-years for short-term and long-term workers, respectively. Like most estimates based on historical data, these exposure estimates are subject to uncertainty. Nevertheless, these results suggest a potential excess risk of death from lung cancer among U.S. workers exposed to the current permissible exposure limit (PEL) for hexavalent chromium of 52 micrograms/m3 because such workers could accumulate exposures (micrograms/m3-years) similar to those associated with excess risk in Hayes et al's cohort. Moreover, many current workers are estimated to be exposed to levels above the PEL. Further exploration of the dose-response relationship for chromium carcinogenesis is indicated.

Air Pollutants, Occupational↗
PubMed1985

The association between dietary sucrose consumption and microbial population shifts at six oral sites in man.

Sucrose-related microbial population shifts were evaluated at 6 oral sites in 22 volunteers, who consumed high-sucrose diets for 21 days followed by low-sucrose diets for 21 days. Culturing was performed at 0, 12, 21, 33 and 42 days of the 6-week experiment. Over 50,000 microbial isolates were characterized and analysed. Analysis of initial cultures showed the following site-specific microbial characteristics of the 6 sites evaluated: (1) molar fissures harboured higher levels of Neisseria species and showed the highest facultative-to-anaerobic ratio; (2) molar fissures and cervical buccal sites showed high Streptococcus sanguis levels and total Gram-positive cocci and fewer Gram-negative bacilli; (3) the tongue and saliva gave high concentrations of Streptococcus salivarius and Veillonella sp. Sucrose intake was positively related to concentrations of yeasts and Streptococcus mutans in the molar fissures; Actinomyces viscosus in the mandibular approximal site; Strep. mutans, Veillonella sp. and Lactobacillus sp. in the maxillary approximal site and Strep. salivarius on the tongue and in saliva. Sucrose intake was negatively related to concentrations of Neisseria sp. on the tongue and total Gram-positive bacilli in saliva. A definite ecological effect of sucrose on the oral microflora was confirmed. The high inter-subject and site variations of target bacteria and the generally low magnitude of shifts, however, discourage implementation of microbiological criteria in dietary assessments.

Bacteria↗