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J M Chen

Publications and source records attributed to J M Chen.

At least 73 records · Page 4Linked to original sources
PubMed2000

High-resolution solution structure of the catalytic fragment of human collagenase-3 (MMP-13) complexed with a hydroxamic acid inhibitor.

The high-resolution solution structure of the catalytic fragment of human collagenase-3 (MMP-13) complexed with a sulfonamide derivative of a hydroxamic acid compound (WAY-151693) has been determined by multidimensional heteronuclear NMR. A total of 30 structures were calculated for residues 7-164 by means of hybrid distance geometry-simulated annealing using a total of 3280 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for the 30 structures is 0.43(+/-0.05) A for the backbone atoms, 0.80(+/-0.09) A for all atoms, and 0.47(+/-0.04) A for all atoms excluding disordered side-chains. The overall structure of MMP-13 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel and anti-parallel arrangement and three alpha-helices where its overall fold is consistent with previously solved MMP structures. A comparison of the NMR structure of MMP-13 with the published 1.6 A resolution X-ray structure indicates that the major differences between the structures is associated with loop dynamics and crystal-packing interactions. The side-chains of some active-site residues for the NMR and X-ray structures of MMP-13 adopt distinct conformations. This is attributed to the presence of unique inhibitors in the two structures that encounter distinct interactions with MMP-13. The major structural difference observed between the MMP-13 and MMP-1 NMR structures is the relative size and shape of the S1' pocket where this pocket is significantly longer for MMP-13, nearly reaching the surface of the protein. Additionally, MMP-1 and MMP-13 exhibit different dynamic properties for the active-site loop and the structural Zn-binding region. The inhibitor WAY-151693 is well defined in the MMP-13 active-site based on a total of 52 distance restraints. The binding motif of WAY-151693 in the MMP-13 complex is consistent with our previously reported MMP-1:CGS-27023A NMR structure and is similar to the MMP-13: RS-130830 X-ray structure.

Amino Acid Sequence↗
PubMed2000

Pressure-induced retention of the lysozyme on reversed-phase liquid chromatography.

This study investigated the effect of pressure on the retention behavior of a model protein, lysozyme, on octadecylsilica (C18) stationary phase under various equilibrium conditions. It is demonstrated that the retention time of the lysozyme was increased by as much as two to three times as the absolute pressure on the viewing window was increased from 23 to 318 bar. This pressure-induced retention was likely to be reversible and the corresponding volume change (deltaV = Vsta - Vmob) was found to be on the order of minus tens to hundreds of mL/mol. Moreover, the pressure-induced retention was also observed for a homologous series of hydrophobic poly-L-phenylalanine, which do not have the secondary structure, and the volume change was determined to be around minus 10 mL/mol per phenylalanine. Perturbations in solute ionization and conformational change are predicted to have a minor impact under the investigated conditions. It is believed that the pressure-induced shift of the equilibria regarding hydrophobic ad-desorption is the major cause of the observed increase of protein retention. About ten phenylalanine-equivalent residues on the lysozyme surface were involved in the hydrophobic association with the chromatographic ligands.

Chromatography, Liquid↗
PubMed2000

Separation of the molecular species of intact phosphatidylethanolamines and their N-monomethyl and N,N-dimethyl derivatives by high-performance liquid chromatography on a C8 column.

We have developed a gradient reversed-phase C8 high-performance liquid chromatography method for the separation of molecular species of phosphatidylethanolamines (PEs) and their N-monomethyl and N,N-dimethyl derivatives. This method uses a 40-min linear gradient of 88-100% methanol, containing ammonium hydroxide as silanol suppressing agent, and is suitable for metabolic studies using both UV detection at 205 nm and radioactivity flow detection. The elution order of a given PE is inversely related to the polarity of its fatty acid constituents. Lipid classes studied here containing the same fatty acyl chains elute in the order: PE-N,N-dimethyl<PE<PE-N-monomethyl<phosphatidylcholine, indicating that elution order is not simply a function of the numbers of methyl groups on the nitrogen atom of PE.

Chromatography, High Pressure Liquid↗
PubMed2000

Identification and characterization of promoters regulating tuf expression in Chlamydia trachomatis serovar F.

Gene expression in the obligate intracellular bacterium Chlamydia trachomatis ranges from nil in the infectious EB form to high in the dividing RB form. Little is known about the mechanisms of gene regulation in chlamydiae and only a few promoter sequences have been characterized. The purpose of our study was to examine the expression of a cluster of genes that are required for translation in C. trachomatis serovar F: infA (encoding Initiation Factor 1), tRNA(Thr), tuf (encoding Elongation Factor Tu), and tRNA(Trp). Primer extension analysis indicated that tuf is expressed in three different mRNAs. Putative promoter sequences for these transcripts were defined as P1 (upstream of tRNA(Thr)), P2 (within infA) and P3 (upstream of infA). Quantitative RT-PCR analysis revealed that P1 transcripts were most abundant at 16 h postinfection (pi), whereas P2 transcripts predominated at 24 h pi. P3 was active at all times pi; however, transcription terminated upstream of tuf at early times pi and continued through tuf at later times. P1 and P3 were active in Escherichia coli, as assessed by CAT expression in promoter-fusion vectors and a chlamydial in vitro transcription system. Site-specific mutagenesis confirmed the importance of the -35 and -10 hexamers in the P1 and P3 promoters. P2 was weakly active in E. coli and inactive in the in vitro transcription system, indicating either that the P2 transcript is processed from a longer transcript or that P2 expression requires a sigma or transcription factor which is not present in E. coli or the in vitro transcription system. Our data suggest that multiple processes play a role in the regulation of tuf gene expression during the developmental cycle.

Amino Acid Sequence↗
PubMed2000

Gene conversion-like missense mutations in the human cationic trypsinogen gene and insights into the molecular evolution of the human trypsinogen family.

Over the past decade, gene conversion has been shown increasingly to be a cause of human disease. Through this process, a functional gene is converted into a mutant by a homologous, nonfunctional one. In this article, we demonstrate that gene conversion is a likely cause of the mutations of the human cationic trypsinogen (PRSS1) gene that are associated with hereditary or sporadic pancreatitis, including the R122H (CGC>CAT: c.365-366 GC>AT), N29I (AAC>ATC: c.86A>T), and A16V (GCC>GTC: c.47C>T) missense mutations. This hypothesis is strongly supported by four lines of observation. First, human group I trypsinogen genes are tandemly repeated and share a high sequence homology between them. Secondly, a possible donor sequence for each variant is present in the PRSS1 gene's paralog(s). Thirdly, there exist uninterrupted sequence tracts ranging from 30 to 114 bp in the putatively converted regions. Finally, Chi-like and palindromic sequences are found in the vicinity of these missense mutations. This theory, if correct, will make the pancreatitis-associated PRSS1 mutations a unique example, as it shows that a functional gene may be converted by several paralogs, and that such an event may even occur between two functional genes (i.e., the N29I mutation), resulting in disease. This adds further to the diversity of genetic mechanisms underlying human disease. In addition, this genetic finding provides, for the first time, concrete evidence of the contribution made by gene conversion to the molecular evolution of the human trypsinogen family.

Base Sequence↗
PubMed2000

Origin and implication of the hereditary pancreatitis-associated N21I mutation in the cationic trypsinogen gene.

The N21I missense mutation in the cationic trypsinogen gene is the second most frequent mutation in hereditary pancreatitis (HP). In this article, we suggest that the N21I mutation most likely arose as a gene conversion event in which the functional anionic trypsinogen gene acted as the donor sequence. This hypothesis is supported by the unique presence of Ile at residue 21 of the anionic gene amongst the several highly homologous trypsinogen genes; a single unbroken tract of nucleotides of up to 113 bp flanking the I21 residue in the anionic trypsinogen gene; and the presence of a chi-like sequence in the 5' proximity and a palindromic sequence in the 3' vicinity of the N21I mutation. Furthermore, a multiple alignment of the partial amino acid sequence of vertebrate trypsins around residue 21 indicated that N21 and I21 may represent advantageously selected mutations of the two functional human trypsinogen genes in evolutionary history. These observations, which are complementary to the previous findings, provide further insights into the genetic mechanism and pathogenic role of the N21I mutation in HP.

Amino Acid Sequence↗
PubMed2000

Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis.

We have examined the role of 2-oleoyl-PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes. In castor microsomal incubation, the label from 14C-oleate of 1-palmitoyl-2-[1-(14)C]oleoyl-sn-glycero-3-phosphoethanolamine is incorporated into TAG containing ricinoleate. The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl-12-hydroxylase using 2-oleoyl-PE as incubation substrate are similar to those for 2-oleoyl-PC (phosphatidylcholine). However, compared to 2-oleoyl-PC, 2-oleoyl-PE is a less efficient incubation substrate of oleoyl-12-hydroxylase in castor microsomes. Unlike 2-oleoyl-PC, 2-oleoyl-PE is not hydroxylated to 2-ricinoleoyl-PE by oleoyl-12-hydroxylase and is not desaturated to 2-linoleoyl-PE by oleoyl-12-desaturase. We have demonstrated the conversion of 2-oleoyl-PE to 2-oleoyl-PC and vice versa. The incorporation of label from 2-[14C]oleoyl-PE into TAG occurs after its conversion to 2-oleoyl-PC, which can then be hydroxylated or desaturated. We detected neither PE-N-monomethyl nor PE-N,N-dimethyl, the intermediates from PE to PC by N-methylation. The conversion of 2-oleoyl-PE to 2-oleoyl-PC likely occurs via hydrolysis to 1,2-diacyl-sn-glycerol by phospholipase C and then by cholinephosphotransferase. This conversion does not appear to play a key role in driving ricinoleate into TAG.

Castor Oil↗
PubMed2000

Identification, using molecular dynamics, of an effector domain of the ras-binding domain of the raf-p74 protein that is uniquely involved in oncogenic ras-p21 signaling.

By comparing the average structures, computed using molecular dynamics, of the ras-binding domain of raf (RBD) bound to activated wild-type ras-p21 and its homologous inhibitory protein, rap-1A, we formerly identified three domains of the RBD that changed conformation between the two complexes, residues 62-76, 97-110, and 111-121. We found that one synthetic peptide, corresponding to RBD residues 97-110, selectively inhibited oncogenic ras-p21-induced oocyte maturation. In this study, we performed molecular dynamics on the Val 12-ras-p21-RBD complex and compared its average structure with that for the wild-type protein. We find that there is a large displacement of a loop involving these residues when the structures of the two complexes are compared. This result corroborates our former finding that the RBD 97-110 peptide inhibits only signal transduction by oncogenic ras-p21 and suggests that oncogenic p21 uses this loop to interact with raf in a unique manner.

Amino Acid Substitution↗
PubMed2000

Molecular basis of hereditary pancreatitis.

Hereditary pancreatitis (HP) is an autosomal dominant disease. Two heterozygous missense mutations, R122H (R117H) and N29I (N21I), in the cationic trypsinogen gene have been clearly associated with HP. The 'self-destruct' model proposed for the R122H mutation is discussed in connection with the existing theory of pancreatitis, and the basic biochemistry and physiology of trypsinogen, with particular reference to R122 as the primary autolysis site of the cationic trypsinogen. Two different genetic mechanisms are identified which cause the R122H mutation, and gene conversion is the likely cause of the N29I mutation. A unifying model, which highlights an indirect impairment on the R122 autolysis site is hypothesised for the N29I mutation. Possible predisposition to pancreatitis by additional DNA variants in the gene, such as the A16V signal peptide cleavage site mutation and the K23R activation peptide cleavage site mutation is suspected, but not proven. Evidence of genetic heterogeneity of HP is reviewed and cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations detected in HP families are re-evaluated. Finally, large scale association studies are expected to clarify the additional variants' role in pancreatitis and to identify new HP genes.

Amino Acid Sequence↗
PubMed2000

Impact of current management practices on early and late death in more than 500 consecutive cardiac transplant recipients.

OBJECTIVE: To study risk factors for early and late death after heart transplantation in the current era. SUMMARY BACKGROUND DATA: The current cardiac transplant population differs from earlier periods in that an increasing number of sicker patients, such as those with ventricular assist device (LVAD) support, prior cardiac allotransplantation, and pulmonary hypertension, are undergoing transplantation. In addition, sensitized patients constitute a greater proportion of the transplanted population. Emphasis has been placed on therapies to prevent early graft loss, such as the use of nitric oxide and improved immunosuppression, in addition to newer therapies. METHODS: Five hundred thirty-six patients undergoing heart transplantation between 1993 and 1999 at a single center were evaluated (464 adults and 72 children; 109 had received prior LVAD support and 24 underwent retransplantation). The mean patient age at transplantation was 44.9 years. Logistic regression and Cox proportional hazard models were used to evaluate the following risk factors on survival: donor and recipient demographics, ischemic time, LVAD, retransplantation, pretransplant pulmonary vascular resistance, and immunologic variables (ABO, HLA matching, and pretransplant anti-HLA antibodies). RESULTS: The rate of early death (less than 30 days) was 8.5% in adults and 8.8% in children. The actuarial survival rate of the 536 patients was 83%, 77%, and 71% at 1, 3, and 5 years, respectively, by Kaplan Meier analysis. Risk factors adversely affecting survival included the year of transplant, donor age, and donor-recipient gender mismatching. Neither early nor late death was influenced by elevated pulmonary vascular resistance, sensitization, prior LVAD support, or prior cardiac allotransplantation. CONCLUSIONS: Previously identified risk factors did not adversely affect short- or long-term survival of heart transplant recipients in the current era. The steady improvement in survival during this period argues that advances in transplantation have offset the increasing acuity of transplant recipients.

Adolescent↗
PubMed2000

Intratympanic gentamicin for the treatment of unilateral Meniere's disease.

OBJECTIVE: To determine the efficacy of intratympanic gentamicin instillation as treatment of incapacitating unilateral Meniere's disease, using a predetermined regimen with a fixed dose. STUDY DESIGN: A prospective study from a single institution between 1988 and 1998. METHODS: One hundred fourteen patients were enrolled in this study. Gentamicin (26.7 mg/mL) was administered three times daily for 4 consecutive days. The Committee on Hearing and Equilibrium Guidelines for Reporting Treatment Results in Meniere's Disease of the American Academy of Otolaryngology and Head and Neck Surgery (1985) were used. RESULTS: Comprehensive data were available for 90 individuals. Complete control of vertigo was achieved in 76 (84.4%), substantial control in 8 (9.0%), limited control in 2 (2.2%), and insignificant control in 4 (4.4%) patients. Disability scores at the end of 2 years were as follows: 76 patients (84.4%) had no disability, 5 (5.6%) had mild disability, 2 (22%) had moderate disability, and 7(7.8%) had severe disability. Caloric testing responses, as determined using electronystagmography, were as follows: 71% of the patients had an absent ice-water response, 16% had a positive ice-water response, and in 13% there continued to be present a bithermal response. Hearing was worse in 22 patients (25.6%), unchanged in 41 (48.2%), and improved in 22 (25.6%). CONCLUSIONS: Intratympanic gentamicin administration using this particular protocol is an effective treatment option for patients with disabling unilateral Meniere's disease. Hearing loss is a distinct possibility, and patients should be advised accordingly.

Adult↗
PubMed2000

Genes, cloned cDNAs, and proteins of human trypsinogens and pancreatitis-associated cationic trypsinogen mutations.

Historically, trypsinogens/trypsins have been one of the most extensively studied enzyme models of protein structure and function. They have received renewed attention after the identification of mutations in the cationic trypsinogen gene as being associated with hereditary pancreatitis. A survey of the literature revealed five cloned cDNAs, but only three protein products of human trypsinogens, and their nomenclature has been confusing. The availability of the complete genomic sequencing of the human trypsinogen gene family made it possible to provide a systematic review of the genes, cDNAs, and protein products of human trypsinogens and to clarify some controversial issues. Further, the confusing coexistence of two systems for naming the cationic trypsinogen mutations is addressed.

Alternative Splicing↗
PubMed2000

Electrocochleography and gentamicin therapy for Ménière's disease: a preliminary report.

OBJECTIVE: It is widely held that an enlarged summating potential (SP) relative to the eighth nerve action potential (AP) is a reflection of endolymphatic hydrops. Aminoglycosides are an accepted treatment for incapacitating Ménière's disease and are known to affect both sensory and secretory cells of the inner ear. The intent of this study was to determine whether this effect on secretory cells could be objectively confirmed by virtue of changes in the electrocochleogram (ECoG) of patients receiving gentamicin therapy for Ménière's disease. STUDY DESIGN: This was a prospective longitudinal study of repeated ECoG measures in three groups of subjects. Ménière's patients undergoing gentamicin treatment were compared with two control groups: individuals with stable Ménière's disease and normal-hearing control subjects. SETTING: The study was conducted at a tertiary referral center. PATIENTS: The sample included 21 normal-hearing subjects, 15 patients with stable unilateral Ménière's disease, and 12 with disabling unilateral Ménière's disease. INTERVENTIONS: For patients with disabling Ménière's disease, gentamicin was administered transtympanically. Audiograms, impedance tests, and ECoG were performed twice for all subjects. MAIN OUTCOME MEASURES: The SP and AP amplitudes, AP latency, and SP/AP ratio of the EcoG were measured. RESULTS: A statistically significant reduction in the SP/AP ratio was observed after gentamicin administration (analysis of variance interaction effect: F2 = 5.64; p = 0.0065). CONCLUSIONS: The significant reduction in the SP/AP ratio in the gentamicin-treated Ménière's group supports the hypothesis that gentamicin improves the electrophysiologic function of the cochlea, possibly by reducing the severity of the associated endolymphatic hydrops.

Acoustic Impedance Tests↗
PubMed2000

Postoperative analgesic requirements in patients undergoing arthroscopic anterior cruciate ligament reconstruction.

Anterior cruciate ligament (ACL) procedures are associated with significant postoperative pain and have traditionally been done on a short-stay hospitalization basis because of concerns for adequate postoperative analgesia. A retrospective chart review was performed to determine postoperative intravenous patient-controlled analgesia (PCA) morphine requirements for 80 patients who had undergone arthroscopically assisted ACL reconstruction under general anesthesia by means of a patellar tendon autograft by 1 of 2 surgeons. The mean +/- SD PCA morphine used after surgery was 20.4+/-20.0 mg. There was a wide interpatient difference in postoperative opioid consumption: the amount of PCA morphine used ranged from 0 mg to 124 mg. A comparison between the surgeons revealed that 1 surgeon had significantly longer intraoperative surgical, tourniquet, and anesthesia times; however, there was no difference in the length of recovery room stay, amount of postoperative PCA morphine used, or time to hospital discharge. Predicting which patients may benefit from short-stay hospitalization after arthroscopic ACL reconstruction may be difficult because of considerable interpatient differences in postoperative analgesic requirements.

Adult↗