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K Chu

Publications and source records attributed to K Chu.

At least 19 recordsLinked to original sources
PubMed1996

A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism.

The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.

Amino Acid Sequence↗
PubMed1996

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.

3T3 Cells↗
PubMed1996

Medicaid managed care: lessons from five states.

With Medicaid managed care enrollment accelerating, policymakers must quickly understand the lessons from the states so that the transition to managed care strengthens access and care systems and avoids problems. Our study of five recent state experiences shows that state objectives must be realistic and well planned. The experience of states with long histories of managed care underscores the conclusion that managed care is not a magic bullet for solving all access and cost concerns and that no amount of managed care can substitute for adequately financed programs that are well understood by beneficiaries, providers, and health plans alike. The results highlight ten lessons from the states.

California↗
PubMed1995

A Fas-associated protein factor, FAF1, potentiates Fas-mediated apoptosis.

Fas, a member of the tumor necrosis factor receptor family, can induce apoptosis when activated by Fas ligand binding or anti-Fas antibody crosslinking. Genetic studies have shown that a defect in Fas-mediated apoptosis resulted in abnormal development and function of the immune system in mice. A point mutation in the cytoplasmic domain of Fas (a single base change from T to A at base 786), replacing isoleucine with asparagine, abolishes the signal transducing property of Fas. Mice homozygous for this mutant allele (lprcg/lprcg mice) develop lymphadenopathy and a lupus-like autoimmune disease. Little is known about the mechanism of signal transduction in Fas-mediated apoptosis. In this study, we used the two-hybrid screen in yeast to isolate a Fas-associated protein factor, FAF1, which specifically interacts with the cytoplasmic domain of wild-type Fas but not the lprcg-mutated Fas protein. This interaction occurs not only in yeast but also in mammalian cells. When transiently expressed in L cells, FAF1 potentiated Fas-induced apoptosis. A search of available DNA and protein sequence data banks did not reveal significant homology between FAF1 and known proteins. Therefore, FAF1 is an unusual protein that binds to the wild type but not the inactive point mutant of Fas. FAF1 potentiates Fas-induced cell killing and is a candidate signal transducing molecule in the regulation of apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Effects of retinoic acid and estrogens on oxytocin gene expression in the rat uterus: in vitro and in vivo studies.

We and others have previously identified functional estrogen (E) and retinoic acid (RA) response elements in the human and rat oxytocin (OT) gene promoters. Whereas there is no direct evidence for a significant role of E or RA in the regulation of rat hypothalamic OT gene expression, we have recently demonstrated that in vivo administration of E strongly stimulates uterine OT gene expression. Here, we show that in vivo administration of RA similarly induces a significant increase in uterine OT gene expression. Moreover, we report that the E and RA effects are reproducible in vitro. Using short-term uterine organ explant cultures derived from 18-day pregnant rats, we found that E (50 nM) and RA (0.4 nM) increased OT mRNA levels 5.2- and 3-fold, respectively, suggesting a direct action of these agents on uterine OT gene expression. Finally, we analyzed uterine E and RA receptor gene expression during pregnancy. Using semi-quantitative Northern blot analysis, we found that mRNAs encoding the E receptor, the RA receptor alpha and RA receptor beta are present in rat uterus and that their levels rise by 3.7-, 3.6- and 5.8-fold, respectively, between day 14 of gestation and term. Taken together, the data suggest that, at term, the rat uterus has an increased capacity to respond to E and RA, and that both agents may be involved in mediating the dramatic increase of OT mRNA accumulation observed in the uterus at term.

Animals↗
PubMed1995

Ligand binding to heme proteins. V. Light-induced relaxation in proximal mutants L89I and H97F of carbonmonoxymyoglobin.

We have studied the proximal mutants L89I and H97F of MbCO with FTIR and temperature-derivative spectroscopy at temperatures between 10 and 160 K. The mutations give rise only to minor alterations of the stretch spectra of the bound and photodissociated CO ligand. The most pronounced difference is a larger population in the A3 substate at approximately 1930 cm-1 in the mutants. The barrier distributions, as determined by temperature-derivative spectroscopy, are very similar to native MbCO after short illumination. Extended illumination leads to substantial increases of the rebinding barriers in native MbCO and the proximal mutants. A larger fraction of light-relaxed states is found in the proximal mutants, implying that the conformational energy landscape has been modified to more easily allow light-induced transitions. These and other spectroscopic data imply that the large changes in the binding properties are brought about by a light-induced conformational relaxation involving the structure at the heme iron. Similarities with spectral hole-burning studies and physical models are discussed.

Animals↗
PubMed1995

Gonadal steroid regulation of oxytocin and oxytocin receptor gene expression.

By Northern blot analysis and in situ hybridization, we have determined that, at term, the rat uterine epithelium represents a major site of oxytocin (OT) gene expression. OT mRNA levels increase > 150-fold during pregnancy and, at term, exceed hypothalamic OT mRNA by a factor of 70. By cryoultramicroscopy, OT immunoreactivity was localized to transport vesicles in the apical compartment of uterine epithelial cells. Estrogens (E) act as a strong inducer of uterine OT gene expression in vivo, and this effect is potentiated 7-fold by concomitant progesterone (P) administration. We have also cloned the rat OT receptor (OTR) gene and developed a polymerase chain reaction (PCR)-based assay to measure OTR mRNA. Whereas OTR mRNA is strongly induced by E, P does not potentiate but slightly attenuates the E-induced rise. However, E-induced OT binding is completely reversed by concomitant P administration, suggesting an additional post-transcriptional effect of P. The mechanisms of E-induction of the uterine OT gene remain unclear, inasmuch as the OTR gene promoter does not contain a classical estrogen response element (ERE). Moreover, transfection analysis of a 3.1 kb OTR gene promoter fragment linked to a luciferase reporter gene indicates that promoter activity is induced 5-fold by calcium ionophore A23187 but not by E.

Animals↗
PubMed1994

Ligand binding to heme proteins: the effect of light on ligand binding in myoglobin.

Extended illumination slows the rebinding of CO to myoglobin after photodissociation at cryogenic temperatures. Two types of models have been put forward to explain the effect: motions of the CO within the heme pocket or conformational transitions of the protein. To resolve this ambiguity, we have studied the effect of extended illumination on ligand binding to horse and sperm whale myoglobin (hMb and swMb) with temperature-derivative spectroscopy, monitoring the reaction in the CO stretch bands in the infrared and the conformation-sensitive band III near 760 nm. The experiments show that the stretch frequency of the photodissociated CO does not change upon illumination, implying that the slowing of the CO rebinding is caused by conformational relaxation of Mb from the bound state toward the deoxy structure. The light-induced relaxation (LIR) depends on the number of photons absorbed but not on the light intensity or duration separately. LIR occurs on photon absorption in either the bound or photodissociated state and depends on the temperature at which the MbCO is illuminated. The LIR proceeds in jumps through a small number of conformational substates. The effective barrier for rebinding increases with each step. The substates populated are similar to those found in the thermally-induced relaxation (TIR) that is observed above 160 K. LIR depends markedly on the structural details; it differs for swMbCO and hMbCO and even for the three A substates of swMbCO. Pronounced differences exist between the effects in MbCO and MbO2. The similarity of LIR and TIR leads to a revised model for ligand binding to swMbCO and hMbCO, in which the relaxation is crucial for the escape of the ligand from the pocket, as was first suggested by Friedman [Friedman, J. M. (1985) Science 228, 1273-1280].

Animals↗
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↗