Biomedical subjects
Publications and source records attributed to D Xie.
Excellent yields and at least 95 % ee can be achieved for the addition of dimethyl malonate to cycloalkenyl acetates by using a palladium complex of the new phosphanyldihydrooxazole ligand L as a catalyst (see scheme). The ligand L can be synthesized from commercially available trans 4-hydroxy-L-proline in four steps. BOC=tert-butoxycarbonyl.
O6-Alkylguanine-DNA alkyltransferase (AGT, EC 2.1.1.63) is a principle DNA repair protein in repairing O6-alkylguanine in DNA, a major premutagenic lesion produced by environmental and therapeutic alkylating agents. AGT plays a critical role in protecting cells against mutation and cytotoxicity induced by these alkylating agents. The existence of a large interindividual variation in human AGT activity level has been observed and we hypothesize that genetic polymorphism of AGT could be an important determinant for this variation. The present study reports the identification of a novel missense polymorphism in the human AGT gene. The polymorphic alteration occurs at codon 143 in exon 5, converting isoleucine (ATC) to valine (GTC). Because Ile143 is adjacent to the alkyl acceptor Cys145 of the AGT active site and is conserved among mammalian AGTs, amino acid substitution at this position may affect the function of AGT. The codon 143 polymorphism appears to be linked to another new polymorphic alteration at codon 178, which converts lysine (AAG) to arginine (AGG). Because it has been reported that human AGT can be truncated at position 176 without loss of activity, the codon 178 polymorphism may not affect AGT activity. The codon 143/178 polymorphism was found in two of 90 (2%) esophageal cancer patients residing in a high incidence area of China, but was not detected in 60 normal individuals residing in the same area. Six of 28 (210%) non-cancer Caucasian individuals, however, were found to carry this polymorphic allele, suggesting a significant ethnic difference in distribution of this codon 143/178 polymorphism between Chinese and Caucasian individuals. In addition, we confirmed the existence of a codon 84 genetic polymorphism previously identified in a Japanese population, which converts leucine (CTT) to phenylalanine (TTT). The distribution of codon 84 polymorphism was 16%, 20% and 36%, respectively, in the Chinese esophageal cancer patients, Chinese and Caucasian non-cancer individuals. Coexistence of codons 84 and 143/178 polymorphic alterations was found in one Caucasian individual. In all the Chinese (n = 150) and Caucasian (n = 28) samples examined, we were unable to detect a previously reported codon 160 polymorphism (Gly to Arg) which occurred in 10-25% of the Japanese individuals and was shown to affect the reaction of AGT with the drug O6-benzylguanine. The functional significance of the codon 143/178 genetic polymorphism of human AGT and its role in determining an individual's susceptibility to environmental alkylating carcinogens and response to alkylating chemotherapeutic drugs both remain to be studied.
The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.
Several immunohistochemical studies showed that p53 protein is expressed in 50 to 80% of esophageal adenocarcinomas (EAs). Mutations of this tumor suppressor gene are present in 40 to 70% of EAs, so it is possible that p53 expression might occur as a result of mechanisms other than gene mutation. The human homologue of the murine double minute-2 gene (mdm-2) is a known regulator of p53 activity, and its expression results in stabilization of the wild-type p53 protein and loss of its tumor suppressor function. In this study, we evaluated the frequency of mdm-2 amplification and expression in EA and investigated the relationship between mdm-2 expression and p53 mutation. Thirty-three resection specimens of EAs and associated Barrett's esophagus were evaluated by immunohistochemical methods for p53 and mdm-2 expression. Sixteen of these cases were also evaluated for p53 mutations with use of polymerase chain reaction, single-strand conformational polymorphism, and DNA sequencing and for mdm-2 amplification with a differential polymerase chain reaction-based amplification analysis. Overexpression of p53 was present in 23 EAs (70%), and 18 EAs (55%) overexpressed mdm-2. p53 mutation was observed in 7 (43%) of 16 cases, whereas mdm-2 gene amplification was not detected in any. To summarize, we found substantial discordance of p53 immunohistochemical features and mutation in EA. Significant expression of mdm-2 occurred only in cases with wild-type p53, whereas all of the cases with p53 mutation showed little if any expression of mdm-2. Also, mdm-2 expression in cases with p53 overexpression but without p53 mutation exceeded mdm-2 expression in cases with p53 overexpression and p53 gene mutation. In cases without p53 mutation, overexpression of mdm-2 occurred in 50% of cases and might be responsible for stabilization of p53 protein and possible loss of tumor suppressor function.
Reported here is our experience with a single or double-face new procedure using a pedicled labial flap for urethral reconstruction in patients treated for extensive urethral damage after obstetrical injury. Between January 1992 to July 1997, 56 cases of urethral damage on African female patients, with an average age of 18 years old, were treated by pedicled labial urethroplasty. This procedure was done by using a single or double-face pedicled flap obtained from the major or minor labia. The flap was then introduced as in a tunnel beneath the vaginal epithelium reaching the damaged urethra. A variety of techniques were proposed: patch for sufficient lengthening (27 cases), tubularized flap allowing complete reconstruction of the urethra (18 cases) and the double-face urethroplasty (11 cases). Good quality urine continence was obtained by using the sub urethral Martius'sling procedure. In 11 cases, we combined the treatment with a colposuspension procedure. The average follow-up was 23 months (ranging from 5 to 47 months). The global success was 82% (52 patients treated). Recovery of normal miction and absence of urinary leak was obtained in 36 cases (69%). While 7 moderate failures occurred (13%), 9 cases were considered complete failures (17%). In view of the high success rate, we consider that the one-stage procedure by the use of a single or double face pedicled labial flap is a choice treatment and highly suitable for the management of extensive urethral cervical damage after obstetrical injury.
To investigate the biochemical properties of the protease encoded by the human endogenous retrovirus, K10 (HERV-K), 213 amino acids of the 3'-end of the HERV-K protease (PR) open reading frame were expressed in Escherichia coli. Autocatalytic cleavage of the expressed polypeptide resulted in an 18.2 kDa protein which was shown to be proteolytically active against a fluorogenic peptide used as a substrate for HIV-1 protease. On the basis of sequence homology and molecular modeling, the 106 N-terminal amino acids of HERV-K PR were predicted to comprise a retroviral protease core domain. An 11.6 kDa protein corresponding to this region was expressed and shown to be a fully functional enzyme. The 11.6 kDa domain of HERV-K PR is unusually stable over a wide pH range, exhibits optimal catalytic activity between pH 4.0 and 5.0, and exists as a dimer at pH 7.0 with a Kd of 50 microM. Like HIV-1 PR, the HERV-K PR core domain is activated by high salt concentrations and processes HIV-1 matrix-capsid polyprotein at the authentic HIV-1 PR recognition site. However, both the 18.2 and 11.6 kDa forms of HERV-K PR were highly resistant to a number of clinically useful HIV-1 PR inhibitors, including ritonavir, indinavir, and saquinavir. This raises the possibility that HERV-K PR may complement HIV-1 PR during infection, and could have implications for protease inhibitor therapy and drug resistance.
Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase. Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein. To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry. The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding. The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses. Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes. Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.
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PURPOSE: To test the hypothesis that lidocaine prolongs the safe period of circulatory arrest during deep hypothermia. METHODS: Sixteen dogs were subjected to cooling, first surface cooling to 30 degrees C and then core cooling to 20 degrees C rectal temperature). The circulation was then stopped for 90 min. In the lidocaine group, 4 mg.kg-1 lidocaine was injected into the oxygenator two minutes before circulatory arrest and 2 mg.kg-1 at the beginning of reperfusion and rewarming. The control group received equivalent volumes of normal saline. Post-operatively, using a neurological deficit scoring system (maximum deficit score-100; minimum-zero indicating that no scored deficit could be detected). Neurological function was evaluated hourly for six hours and then daily for one week, the pharmacokinetic parameters were calculated using one compartment model. RESULTS: On the seventh day, the neurological deficit score and overall performance were better in the lidocaine (0.83 +/- 2.04) than in the control group (8.33 +/- 4.08 P < 0.05). During the experiment, the base excess values were also better in the lidocaine than in the control group (at 30 min reperfusion: -4.24 +/- 1.30 vs -8.20 +/- 2.82 P < 0.01, at 60 min reperfusion was -3.34 +/- 1.87 vs -7.52 +/- 2.40 (P < 0.01). On the eighth day the extent of pathological changes were milder in the lidocaine group than that in the control group. The elimination half life of lidocaine was 40.44 +/- 7.99 during hypothermia and 2.01 +/- 4.56 during rewarming. CONCLUSIONS: In dogs lidocaine prolongs the safe duration of circulatory arrest during hypothermia.
The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.
Tumor suppressor protein p53 is a tetrameric phosphoprotein that activates transcription from several cell cycle regulating genes in response to DNA damage. Tetramer formation is critical to p53's ability to activate transcription; however, posttranslational modifications and protein stabilization also contribute to p53's ability to activate transcription. To determine if phosphorylation affects tetramer formation, we synthesized phosphopeptides corresponding to residues 303-393 of human p53, which includes the domain responsible for tetramer formation. Phosphate was chemically incorporated at Ser315, Ser378, or Ser392 and also at both Ser315 and Ser392. Equilibrium ultracentrifugal analyses showed that phosphorylation at Ser392 increased the association constant for reversible tetramer formation nearly 10-fold. Phosphorylation of either Ser315 or Ser378 had little effect on tetramer formation, but phosphorylation of Ser315 largely reversed the effect of phosphorylation at Ser392. Analyses by calorimetry demonstrated that phosphorylation may influence subunit affinity (and, in turn, DNA binding) by an enthalpy-driven process, possibly between the C-terminal residues and the region immediately adjacent to Ser315. The Kd for the tetramer-monomer transition of the unphosphorylated p53 C-terminal domain was determined to be approximately 1-10 microM. Thus, in normal, undamaged cells p53 may be largely monomeric. Enhancement of tetramer formation through phosphorylation of Ser392, coupled with a DNA-damage-induced increase in its nuclear concentration, could provide a switch that activates p53 as a transcription factor in response to DNA damage.
Methods of separating N-acetyl-1,6-diaminohexane (NADAH) and its immobilization to diol-silica have been developed. Hexamethylene bisacetamide (HMBA) and its metabolite NADAH are used as inducers of leukemia cell differentiation. The inducing mechanism of HMBA is still not clear. Experiments show that HMBA and NADAH undergo relatively strong hydrophobic reactions and do not readily undergo ion-exchange with the proteins of the cytosolic fraction of HL-60 cells during immobilization of NADAH; the retention time of the proteins was longer than that of the phosphatides. These results show that the adsorption of HMBA and NADAH to proteins was higher than that to phosphatides. The expected biospecific receptor binding with HMBA has not been found.
Human tumor suppressor protein p53 is a 393-amino acid phosphoprotein that enhances transcription in response to DNA damage from several genes that regulate cell cycle progression. The tetrameric state of p53 is critical to wild-type function; the p53 tetramerization element is located in the C-terminal region of the protein. This region is phosphorylated at several evolutionarily conserved serines, suggesting that phosphorylation may be an important regulator of p53 function. In order to determine the effect of phosphorylation on tetramer formation, we synthesized phosphopeptides corresponding to p53(Ser303-Asp393) with phosphate incorporated at Ser315, Ser378, or Ser392, and at both Ser315 and Ser392. Equilibrium ultracentrifugation analysis showed that phosphorylation at Ser392 increased the association constant for tetramer formation nearly ten-fold. By itself, phosphorylation at Ser315 or Ser378 had little effect on tetramer formation, but Ser315 largely reversed the effect of phosphorylation at Ser392. Analysis by calorimetry suggests that phosphorylation may influence subunit affinity by an enthalpy driven process.
The human embryonic lung fibroblasts transformation was induced by chemical mutant Glycidyl Methacrylate (GMA) in vitro. Transformed clonies were isolated and then exon 5 and exon 8 of p53 gene were specifically amplified by polymerase chain reaction. Amplified fragments were detected by using silver stained single strand conformation polymorphism analysis. The results showed that exon 8 altered in the transformed cells, indicating that p53 gene played an important role in the human embryonic lung fibroblasts transformation induced by GMA.
OBJECTIVE: To explore the effect of cell components in giant cell tumor of bone (GCT) and cytokines expressed by them on osteolysis. METHODS: Mononuclear stromal cells and multinucleated giant cells (MGCs) were isolated from 10 cases of GCT, and fibroblast-like stromal cells were obtained by long-term culture of mononuclear stromal cells. Osteolytic capability of above isolated cells were tested in an in vitro cell-bone resorption model. RESULTS: All cell components isolated from GCT had capability to resorb bone matrix directly. Exogenous tumore necrosis factor-alpha (TNF-alpha) could significantly increase the bone resorption induced by both kinds of stromal cell. There were higher level of TNF-alpha and greater expression rate of macrophage colony stimulating factor (M-CSF) in GCT than in the osteosarcoma tissues or the normal serum. CONCLUSIONS: The characteristic bone resorption behavior of GCT might be conducted by all it's three major cell components, and this bone resorption process could be promoted by both TNF-alpha and M-CSF they expressed.
The expression profile of the mouse Adh-1 gene, which encodes class I alcohol dehydrogenase enzyme (ADH), is complex and includes tissue specificity and differential hormone responsiveness. Whereas kidney Adh-1 transcription rate is stimulated six- to sevenfold by testosterone treatment, adrenal gland ADH-1 mRNA is reduced to less than 5% of control level within 18 h following hormone administration. Androgen receptor is required for both responses since neither occurs in Tfm mutant mice lacking receptor. Hormonal and tissue-specific aspects of Adh-1 regulation were studied in transgenic mice harboring either of two constructs containing either -2.5 kb or -10 kb of 5'-flanking sequence attached to an Adh-1 minigene. The minigene transcript was expressed in kidney and adrenal tissues, but not liver, in five independent lines harboring a transgene with -2.5 kb of 5'-flanking sequence. Androgen treatment repressed the level of the minigene transcript in adrenal gland, but did not cause induction in kidney. In four lines of transgenic mice carrying the construct with -10 kb of 5'-flanking sequence, the minigene transcript was both repressed in adrenal and induced in kidney by testosterone. These lines have no detectable transgene expression in liver tissue. The -10 kb region in the mouse Adh-1 gene contains necessary controlling regions for proper tissue expression and hormonal regulation in kidney and adrenal; however, this region does not contain all essential elements necessary for expression in liver.
OBJECTIVES: The objectives of this study were (1) to develop polyacid formulations through the incorporation of amino acid-derived monomers with carboxylic acid groups at various distances away from the polymer backbone to allow for greater flexibility, less rigid ionic cluster formation and improved solubility, and (2) to test selected physical and handling properties of experimental ionomers with a conventional glass ionomer as a control. METHODS: The polycarboxylic acids prepared and used in glass ionomer formulation in this study included N-acryloylglutamic acid (AGA) and N-acryloyl-6-aminocaproic acid (AACA)- modified acrylic acid- ++itaconic acid copolymers, where the acrylic acid:itaconic acid:amino acid monomers were combined in different proportions. The characterization and purity of the monomers were determined by FTIR and their melting points. The characterization of synthesized polymers included molecular weight and relative viscosity determinations. The compressive strengths, diametral tensile strengths, flexural strengths and fracture toughness of the experimental ionomers and a commercially available ionomer (control) were measured after storage in water, at 37 degrees C for 1 h or 7 d. The working times and setting times of the experimental ionomers were compared to the control specimens. Separate analysis of variance and Tukey's tests were used to study the statistical significance of the physical strength parameters as a function of materials and storage times. RESULTS: Significant increases (p< 0.001) in diametral tensile, compressive, flexural strengths and fracture toughness were observed in the AGA co-polymers, while significant increases were observed in diametral and flexural strengths in the AACA co-polymers compared to the control Fuji II. The working and setting times of all except one experimental ionomer studied were comparable to the controls. SIGNIFICANCE: The use of amino acid-modified acrylic monomers to produce water soluble copolymers of acrylic-itaconic acid offers a new route of discovery to produce chemical-cured glass ionomers with improved physical properties. The spacer chain length, the hydrophobicity of the chains, the molecular weight and viscosity of the polymer all played important roles in determining the physical properties of the material.
In this paper, we extend the Hierarchical Mixture of Experts (HME) to temporal processing and explore it for a substantial problem, that of text-dependent speaker identification. For a specific multiway classification, we propose a generalized Bernoulli density instead of the multinomial logit density to avoid the instability during training. Time-delay technique is applied for spatio-temporal processing in the HME and a combining scheme is presented for combining multiple time-delay HMEs in order to complete a multi-scale analysis for the temporal data. Using the time-delay HME along with the EM algorithm as well as the combination of multiple time-delay HMEs, the speaker identification system has a good performance and yields significantly fast training. We have also addressed some issues about the time-delay techniques in the HME.