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F Wu

Publications and source records attributed to F Wu.

At least 19 recordsLinked to original sources
PubMed2001

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase.

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.

Amino Acid Substitution↗
PubMed2001

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues.

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.

Amino Acid Substitution↗
PubMed2001

YY1 as a regulator of replication-dependent hamster histone H3.2 promoter and an interactive partner of AP-2.

In analyzing cis-regulatory elements important for cell cycle control of the replication-dependent hamster histone H3.2 gene, we discovered a binding site for the transcription factor YY1 embedded within GC-rich sequences between the two tandem CCAAT repeats proximal to the TATA element. Base mutations that specifically eliminated YY1 binding resulted in suppression of the S phase induction of the H3.2 promoter. In addition, we discovered that YY1 is an interactive partner of AP-2, which also binds the H3.2 promoter and regulates its cell cycle-dependent expression. The critical domains for YY1 and AP-2A interaction are mapped, revealing that the N-terminal portion of YY1 (amino acids 1-300) and the DNA-binding/dimerization region of AP-2A are required. Our results suggest that YY1, acting as a transcription factor binding to its site on the promoter, or through protein-protein interaction with AP-2, may be part of a regulatory network including key cell cycle regulators such as c-Myc and Rb in controlling growth- and differentiation-regulated gene expression.

Animals↗
PubMed2001

Rapid bidirectional modulation of mRNA expression and export accompany long-term facilitation and depression of Aplysia synapses.

Serotonin (5-HT) and the neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) modulate synaptic efficacy of sensory neurons (SNs) of Aplysia in opposite directions and for long duration. Both long-term responses require changes in mRNA and protein synthesis. The SN-specific neuropeptide, sensorin A, is a gene product that appears to be increased by 5-HT and decreased by FMRFa. We examined whether changes in sensorin A mRNA levels in the cell body and neurites of SNs accompany long-term facilitation and depression. Both 5-HT and FMRFa evoked rapid changes in sensorin A mRNA levels in the SN cell bodies: an increase with 5-HT and a decrease with FMRFa. Parallel changes in sensorin A mRNA levels in SN neurites were detected 2 h and 4 h later. These rapid changes in mRNA expression and net export required the presence of the appropriate target motor cell L7. The neuromodulators failed to produce changes in mRNA expression or export when SNs were cultured alone or with the inappropriate target cell L11. The changes in mRNA expression were transient because mRNA levels returned to control values 24 h after treatment, while synaptic efficacy remained altered by the respective treatments. These results indicate that two neuromodulators produce distinct, but transient, target-dependent effects on expression and export of a cell-specific mRNA that correlate with changes in synaptic plasticity.

Animals↗
PubMed2001

Analysis of 76Br-BrdU in DNA of brain tumors after a PET study does not support its use as a proliferation marker.

76Br-bromodeoxyuridine has previously been suggested as a PET tracer to characterize proliferation potential. However, in animal studies a large fraction of the tissue radioactivity is due to 76Br-bromide, which remains extracellular for extensive periods and contributes significantly to the level of radioactivity. The present project aimed at investigating whether in human brain tumors, sufficient amounts of 76Br-bromodeoxyuridine would be incorporated into DNA, to motivate further attempts with this tracer. Eight patients with brain tumors: 3 meningiomas, 2 astrocytoma grade IV, 1 astrocytoma oligodendroglioma grade II-IV and 2 metastases, were examined with PET and 76Br-BrdU on three occasions: immediately after injection of the tracer, at 4-6, and at 18-20 hours after administration. After the first PET study, diuresis was introduced and maintained for about 12 hours. About 20 hours after tracer administration, 200 mg/m(2) bromodeoxyuridine was administered to 7 patients median 5.8 (range 1-22) hours prior to operation allowing the immunohistochemical analysis of the proliferation potential. During the operation, tumor samples were taken and radioactivity in DNA extracted and measured. The uptake of radioactivity was higher in the tumors than in brain parenchyma. However, in the operative samples only 1-27% (average: 9%) of the radioactivity was found in the DNA fraction. The plasma radioactivity remained high throughout the study with only minimal signs of elimination by the diuresis. 76Br-BrdU is extensively metabolized to 76Br-bromide, and only a minor fraction of the radioactivity is found in the DNA fraction, making it unlikely that this tracer can be used for assessment of proliferation potential.

Aged↗
PubMed2001

Determinants of sex hormone-binding globulin in normal postmenopausal women.

OBJECTIVE: To examine the factors influencing the levels of sex hormone-binding globulin (SHBG) in normal postmenopausal women by assessing the relationship between SHBG and measured anthropometric, metabolic and hormonal variables. DESIGN: Cross-sectional, observational study. SUBJECTS AND METHODS: Seventy normal postmenopausal women aged 47-71 years (mean 58 years), participated in the study. Information was collected on medical, reproductive and smoking history, alcohol use, dietary intake and physical activity. Body composition measurements using dual-energy absorptiometry, and analyses of biochemical and hormonal indices were performed. RESULTS: Bivariate correlation coefficients indicated that SHBG was inversely related to body weight (r = - 0.44), fat mass (r = - 0.35), and abdominal obesity (r = - 0.42). It was also inversely related to the glucose and insulin levels during an oral glucose tolerance test (- 0.24 < r < - 0.40), serum oestradiol (r = - 0.26), and physical activity (r = - 0.24). Multiple regression analysis indicated that significant independent correlates of SHBG concentration were fat mass, physical activity, alcohol intake, serum oestradiol, and insulin-like growth factor-1, all having a negative impact on SHBG. CONCLUSIONS: From these observed associations, it is concluded that maintenance of body weight, moderate alcohol consumption, and physical activity will tend to reduce SHBG concentrations in postmenopausal women, thereby increasing the levels of free oestradiol. This mechanism could mediate the beneficial effects of these factors in preventing the development of osteoporosis and cardiovascular disease.

Aged↗
PubMed2000

Cell-specific changes in expression of mRNAs encoding splice variants of aplysia cell adhesion molecule accompany long-term synaptic plasticity.

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long-term facilitation (LTF) of SN synapses evoked by serotonin (5-HT) or in the motor neuron L7 during the early phases of long-term depression (LTD) of SN synapses evoked by Phe-Met-Arg-Phe-amide (FMRFa). We used single cell RT-PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5-HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI-linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5-HT evoked changes that exaggerated cell-specific differences in isoform expression. FMRFa evoked changes that reduced cell-specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell-specific regulation of splice variant expression may contribute to different forms of long-term synaptic plasticity.

Alternative Splicing↗
PubMed2000

Capillary electrophoretic separation of the enantiomers of organophosphates with a phosphorus stereogenic center using the sodium salt of octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin as resolving agent.

The sodium salt of the single-isomer, chiral resolving agent, octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin (ODAS-gammaCD) has been used for the capillary electrophoretic separation of the enantiomers of alkylarylphosphates which carry a phosphorus-based stereogenic center. The effective mobilities and separation selectivities were measured at different ODAS-gammaCD and methanol concentrations to find the conditions under which the minor enantiomers could be adequately quantitated in samples obtained by chemical resolution of the racemic mixtures. This work extends the utility of ODAS-gammaCD to a hitherto unexplored field, the capillary electrophoretic separation of the enantiomers of organophosphorus compounds.

Cyclodextrins↗
PubMed2000

Interactions of the HIV-1 fusion peptide with large unilamellar vesicles and monolayers. A cryo-TEM and spectroscopic study.

We have examined the interaction of the human immunodeficiency virustype 1 fusion peptide (23 amino acid residues) and of a Trp-containing analog with vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Both the native and the Trp-substituted peptides bound the vesicles to the same extent and induced intervesicular lipid mixing with comparable efficiency. Infrared reflection-absorption spectroscopy data are compatible with the adoption by the peptide of a main beta-sheet structure in a cospread lipid/peptide monolayer. Cryo-transmission electron microscopy observations of peptide-treated vesicles reveal the existence of a peculiar morphology consisting of membrane tubular elongations protruding from single vesicles. Tryptophan fluorescence quenching by brominated phospholipids and by water-soluble acrylamide further indicated that the peptide penetrated into the acyl chain region closer to the interface rather than into the bilayer core. We conclude that the differential partition and shallow penetration of the fusion peptide into the outer monolayer of a surface-constrained bilayer may account for the detected morphological effects. Such single monolayer-restricted interaction and its structural consequences are compatible with specific predictions of current theories on viral fusion.

Acrylamide↗
PubMed2000

Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme.

Atrial natriuretic peptide (ANP) is a cardiac hormone essential for the regulation of blood pressure. In cardiac myocytes, ANP is synthesized as a precursor, pro-ANP, that is converted to biologically active ANP by an unknown membrane-associated protease. Recently, we cloned a transmembrane serine protease, corin, that is highly expressed in the heart. In this study, we examine effects of corin on pro-ANP processing. Our results show that recombinant human corin converts pro-ANP to ANP and that the cleavage in pro-ANP by corin is highly sequence specific. Our findings suggest that corin is the long-sought pro-ANP-converting enzyme and that the corin-mediated pro-ANP activation may play a role in regulating blood pressure.

Animals↗
PubMed2000

Presynaptic morphological changes associated with long-term synaptic facilitation are triggered by actin polymerization at preexisting varicositis.

Morphological changes are thought to contribute to the expression of long-term synaptic plasticity, a cellular basis for learning and memory. The mechanisms mediating the initiation and maintenance of the morphological changes are poorly understood. We repeatedly imaged the axonal arbors of mechanosensory neurons of Aplysia as they formed new synaptic varicosities and axonal branches after applications of serotonin that cause long-term synaptic facilitation. New varicosities formed exclusively from preexisting varicosities, by splitting or branch outgrowth. These changes were prevented by cytochalasin D, which blocks actin polymerization and the turnover of actin filaments. The suppression of the morphological changes by cytochalasin D did not impair their expression when cytochalasin D was removed 24 hr after exposure to serotonin. These results imply that serotonin induces persistent effects at preexisting presynaptic varicosities, which enhance actin polymerization, and that this is essential for presynaptic morphological changes of long-term facilitation.

Actins↗
PubMed2000

Activation of the cytoplasmic c-Abl tyrosine kinase by reactive oxygen species.

The ubiquitously expressed c-Abl protein tyrosine kinase localizes to both the nucleus and cytoplasm. The nuclear form of c-Abl is activated in the cellular response to genotoxic stress. Here we show that cytoplasmic c-Abl is activated by oxidative stress. The results also demonstrate that mitochondrial cytochrome c is released in the cellular response to H(2)O(2) and that this effect is mediated by a c-Abl-dependent mechanism. In concert with these results, we show that H(2)O(2)-induced apoptosis is attenuated in c-Abl-deficient cells. These findings demonstrate that cytoplasmic c-Abl is involved in the apoptotic response of cells to oxidative stress.

Acetylcysteine↗
PubMed2000

Interaction between protein kinase C delta and the c-Abl tyrosine kinase in the cellular response to oxidative stress.

Protein kinase C (PKC) isoforms are phosphorylated on tyrosine in the response of cells to oxidative stress. The present studies demonstrate that treatment of cells with hydrogen peroxide (H(2)O(2)) induces binding of the PKCdelta isoform and the c-Abl protein-tyrosine kinase. The results show that c-Abl phosphorylates PKCdelta in the H(2)O(2) response. We also show that PKCdelta phosphorylates and activates c-Abl in vitro. In cells, induction of c-Abl activity by H(2)O(2) is attenuated by the PKCdelta inhibitor, rottlerin, and by overexpression of the regulatory domain of PKCdelta. These findings support a functional interaction between PKCdelta and c-Abl in the cellular response to oxidative stress.

Animals↗
PubMed2000

Distribution of (76)Br-labeled antisense oligonucleotides of different length determined ex vivo in rats.

Oligonucleotides may hybridize with high selectivity to an RNA sequence and can be used for the monitoring of gene expression or for its inhibition in experimental or therapeutic purposes. As part of the development of positron emission tomography (PET) methods, different lengths (30, 20, 12 and 6 mer) of antisense phosphorothioate oligonucleotides complementary to rat chromogranin A were labeled with [(76)Br] using a prosthetic group. The (76)Br-oligonucleotides were injected into rat's tail vein (1-2 MBq/rat), and the radioactivity distribution was analyzed after 20 h using whole body autoradiography or by measurement of organ radioactivity concentration. The whole body autoradiography showed different distribution depending on the oligonucleotide length. The organs with highest uptake changed from kidney cortex (with 6 or 12 mer), kidney cortex and liver (with 20 mer), to liver and spleen (with 30 mer). With 20 or 30 mer sequences, uptake could be observed in the adrenals. Kidneys and livers from rats receiving 20 mer or 30 mer (76)Br-oligonucleotides were analyzed with respect to subcellular distribution and DNA/RNA/protein fraction. 30%-45% of the radioactivity was found in the nuclear fraction. More than 80% of the radioactivity was recovered in the high molecular weight fraction (as proteins or oligonucleotides longer than 10 mer) using size exclusion (NAP 5) gelfiltration or cetylpyridinium bromide (CPB) precipitation. This work indicates the potential to perform kinetic whole body studies of (76)Br-oligonucleotides using PET.

Animals↗
PubMed2000

Dynamical simulation of LACBED patterns in cross-sectioned heterostructures

The Large Angle Convergent Beam Electron Diffraction (LACBED) technique has been applied to determination of the tetragonal mismatch in coherent Si/Si1-xGex/Si heterostructures. Two-dimensional (2D) dynamical simulation of the LACBED patterns has been performed and compared with the corresponding experimental ones. A good agreement is found in the whole simulated area, particularly as regards the splitting of the Bragg contours, due to the strain field present in the TEM cross-sections.

Journal Article↗
PubMed2000

Control of arterial blood pressure and renal sodium excretion by nitric oxide synthase in the renal medulla.

Work in our laboratory has focused on the role of nitric oxide synthase (NOS) in the regulation of renal medullary function. Biochemical studies demonstrated that the renal medulla is enriched in immunoreactive NOS protein and NOS enzymatic activity when compared with the renal cortex. Further experiments showed large amounts of NOS activity in the inner medullary collecting ducts, while moderate NOS activity was found in glomeruli and vasa recta and minimal NOS activity was detected in other nephron segments examined. In subsequent functional studies, selective renal medullary infusion of NOS stimulators (bradykinin or acetylcholine) or inhibitors (L-NAME) preferentially altered medullary blood flow. The alterations in medullary flow were associated with parallel changes in sodium and water excretion. Similar to the effects observed in anaesthetized rats, chronic infusion of L-NAME directly into the renal medullary interstitial space of conscious, uninephrectomized SD rats selectively decreased renal medullary blood flow throughout a 5-day L-NAME infusion period. The decrease in medullary blood flow was associated with retention of sodium and the development of hypertension, and the effects were reversible. In contrast to the effects of chronic NOS inhibition, renal medullary infusion of NOS substrate L-arginine prevented the development of sodium-sensitive hypertension in the Dahl salt-sensitive rat placed on a high sodium diet. The data reviewed in this paper indicate that NOS isoforms expressed in the renal medulla have a potent influence on renal medullary tubular and vascular function with consequential effects on fluid and electrolyte homeostasis and arterial blood pressure.

Animals↗
PubMed2000

Tubal sterilization and use of an IUD and risk of cervical cancer.

The relationship of tubal sterilization and use of an intrauterine device (IUD) to the risk of invasive squamous cell cervical cancer was evaluated in a case-control study carried out in Shandong, China, from 1989 to 1991. Patients (cases) were 272 women aged 30-77 years with newly diagnosed invasive squamous cell cervical cancer in Shandong Province Tumor Hospital. Controls were 893 randomly selected screened women matched to the cases by age (within 2 years) and county. A decrease in risk was observed in uses of an IUD, especially in long-term users under age 33. A nonsignificant decrease in risk in women under age 33 who had had a tubal ligation was also observed, especially in the first 10 years since the procedure. Tubal ligation and IUD insertion and removal provide opportunities to screen women for cervical cancer and reduce the risk of invasive disease.

Adult↗