Biomedical subjects

Yan Feng

Publications and source records attributed to Yan Feng.

At least 73 records · Page 4Linked to original sources

PubMed2004

Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1.

Acylpeptide hydrolases (APH; also known as acylamino acid releasing enzyme) catalyze the removal of an N-acylated amino acid from blocked peptides. The crystal structure of an APH from the thermophilic archaeon Aeropyrum pernix K1 to 2.1 A resolution confirms it to be a member of the prolyl oligopeptidase family of serine proteases. The structure of apAPH is a symmetric homodimer with each subunit comprised of two domains. The N-terminal domain is a regular seven-bladed beta-propeller, while the C-terminal domain has a canonical alpha/beta hydrolase fold and includes the active site and a conserved Ser445-Asp524-His556 catalytic triad. The complex structure of apAPH with an organophosphorus substrate, p-nitrophenyl phosphate, has also been determined. The complex structure unambiguously maps out the substrate binding pocket and provides a basis for substrate recognition by apAPH. A conserved mechanism for protein degradation from archaea to mammals is suggested by the structural features of apAPH.

Aeropyrum↗

PubMed2004

Crystal structure of the hyperthermophilic inorganic pyrophosphatase from the archaeon Pyrococcus horikoshii.

A homolog to the eubacteria inorganic pyrophosphatase (PPase, EC 3.6.1.1) was found in the genome of the hyperthermophilic archaeon Pyrococcus horikoshii. This inorganic pyrophosphatase (Pho-PPase) grows optimally at 88 degrees C. To understand the structural basis for the thermostability of Pho-PPase, we have determined the crystal structure to 2.66 A resolution. The crystallographic asymmetric unit contains three monomers related by approximate threefold symmetry, and a hexamer is built up by twofold crystallographic symmetry. The main-chain fold of Pho-PPase is almost identical to that of the known crystal structure of the model from Sulfolobus acidocaldarius. A detailed comparison of the crystal structure of Pho-PPase with related structures from S. acidocaldarius, Thermus thermophilus, and Escherichia coli shows significant differences that may account for the difference in their thermostabilities. A reduction in thermolabile residues, additional aromatic residues, and more intimate association between subunits all contribute to the larger thermophilicity of Pho-PPase. In particular, deletions in two loops surrounding the active site help to stabilize its conformation, while ion-pair networks unique to Pho-PPase are located in the active site and near the C-terminus. The identification of structural features that make PPases more adaptable to extreme temperature should prove helpful for future biotechnology applications.

Amino Acid Sequence↗

PubMed2004

The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing.

Resealing after wounding, the process of repair following plasma membrane damage, requires exocytosis. Vacuolins are molecules that induce rapid formation of large, swollen structures derived from endosomes and lysosomes by homotypic fusion combined with uncontrolled fusion of the inner and limiting membranes of these organelles. Vacuolin-1, the most potent compound, blocks the Ca(2+)-dependent exocytosis of lysosomes induced by ionomycin or plasma membrane wounding, without affecting the process of resealing. In contrast, other cell structures and membrane trafficking functions including exocytosis of enlargeosomes are unaffected. Because cells heal normally in the presence of vacuolin-1, we suggest that lysosomes are dispensable for resealing.

Animals↗

PubMed2004

Morphology of cultured human epidermal melanocytes observed by atomic force microscopy.

The objective of this study was to image the surface structure of cultured human epidermal melanocytes using atomic force microscopy (AFM). Epidermis obtained from human foreskins was treated with 0.5% dispase. Cell suspensions of the epidermis were prepared and seeded in six-well plates, in which sheets of mica had been placed. Samples for AFM were fixed on mica and scanning AFM images were captured by contacting and tapping modes operated under normal atmospheric pressure and temperature. Human epidermal melanocytes exhibited rounded, oval, triangular or quadrangular perikarya from which eight to 10 thick dendrites arose. These dendrites first bifurcated near the soma and then divided profusely into daughter branches, which spread out in all directions. We observed string-like long thin projections, growth cones and shorter thicker projections, which arose from the dendritic shafts, in which groups of melanosomes were arrayed. In addition to such structures, the most striking feature was the presence of filopodia arising from the melanocyte dendrite tips and the melanocyte cell body, many of which contained melanosomes. The termini of dendrites formed unbranched terminal protrusions (approximately 1,500-2,000 nm wide) consisting of two to three melanosomes wrapped in an arc, with their filopodia extending outwards. The tips of these structures also appeared to be squeezed and finally pinched off by the melanocyte to form a pouch filled with numerous melanosomes. We conclude that secondary and tertiary branches and subordinate branches might take part in transferring melanosomes into keratinocytes in addition to the transfer through the tips of the dendritic shafts. The melanin granules were expelled by exocytosis.

Cells, Cultured↗

PubMed2004

Beta2 adrenergic receptor gene Arg16Gly polymorphism is associated with therapeutic efficacy of benazepril on essential hypertension in Chinese.

BACKGROUND: There is considerable variability in individual response to antihypertensive agents. The reason for this is not known, but may be related to individual genetic variability. This study examined whether the therapeutic efficacy of benazepril on essential hypertension is modified by beta2 adrenergic receptor gene (ADRB2) Arg16Gly (R16G) polymorphism. METHODS AND RESULTS: We conducted a family-based study of 321 and 610 hypertensive subjects from Yuexi and Huoqiu Counties of Anhui, China, respectively. Both systolic and diastolic blood pressures (SBP and DBP) before and after a 15-day benazepril treatment were measured. ADRB2 R16G genotypes were determined for all subjects. ADRB2 G16 allele frequency was found to be 41.0% and. 47.4% in Huoqiu and Yuexi, respectively. In Yuexi family-based association test (FBAT) revealed that the G16 allele was associated with a greater DBP decrease in response to a 15-day benazepril treatment (Z = 2.12, P = 0.03), and the data were consistent with a dominant inheritance model. A similar trend was observed in Huoqiu Chinese, but the magnitudes of effects were smaller and did not reach statistical significance. The FBAT results were further confirmed by using a generalized estimating equation model. CONCLUSION: Our family-based study provided the first evidence that ADRB2 R16G polymorphism may play an important role in DBP response to benazepril treatment, although the magnitude of the effect appears to be modified by other risk factors such as plasma lipid and glucose profiles.

Adult↗

PubMed2004

Chemical genetic screening identifies sulfonamides that raise organellar pH and interfere with membrane traffic.

Chemical genetics seeks to identify small molecules that afford functional dissection of cell biological pathways. Previous screens for small molecule inhibitors of exocytic membrane traffic yielded the identification and characterization of several compounds that block traffic from the Golgi to the cell surface as well as transport from the endoplasmic reticulum to the Golgi network [Feng et al. Proc Natl Acad Sci USA 2003;100:6469-6474; Yarrow et al. Comb Chem High Throughput Screen 2003;6:279-286; Feng et al. EMBO Reports 2004: in press]. Here, we screened these inhibitors for potential effects on endocytic membrane traffic. Two structurally related sulfonamides were found to be potent and reversible inhibitors of transferrin-mediated iron uptake. These inhibitors do not block endoplasmic reticulum-to-Golgi transport, but do disrupt Golgi-to-cell surface traffic. The compounds are members of a novel class of sulfonamides that elevate endosomal and lysosomal pH, down-regulate cell surface receptors, and impair recycling of internalized transferrin receptors to the plasma membrane. In vitro experiments revealed that the sulfonamides directly inhibit adenosine triphosphate (ATP) hydrolysis by the V-ATPase and that they also possess a potent proton ionophore activity. While maintenance of organellar pH is known to be a critical factor in both endocytosis and exocytosis, the precise role of acidification, beyond the uncoupling of ligands from their receptors, remains largely unknown. Identification of this novel class of sulfonamide inhibitors provides new chemical tools to better understand the function of organelle pH in membrane traffic and the activity of V-ATPases in particular.

Adenosine Triphosphatases↗

PubMed2004

Utility of sparse concentration sampling for citalopram in elderly clinical trial subjects.

The objective of this study was to evaluate whether the disposition of the selective serotonin reuptake inhibitor, citalopram, could be robustly captured using 1 to 2 concentration samples per subject in 106 patients participating in 2 clinical trials. Nonlinear mixed-effects modeling was used to evaluate the pharmacokinetic parameters describing citalopram's disposition. Both a prior established 2-compartment model and a de novo 1-compartment pharmacokinetic model were used. Covariates assessed were concomitant medications, race, sex, age (22-93 years), and weight. Covariates affecting disposition were assessed separately and then combined in a stepwise manner. Pharmacokinetic characteristics of citalopram were well captured using this sparse sampling design. Two covariates (age and weight) had a significant effect on the clearance and volume of distribution in both the 1- and 2-compartment pharmacokinetic models. Clearance decreased 0.23 L/h for every year of age and increased 0.14 L/h per kilogram body weight. It was concluded that hyper-sparse sampling designs are adequate to support population pharmacokinetic analysis in clinically treated populations. This is particularly valuable for populations such as the elderly, who are not typically available for pharmacokinetic studies.

Administration, Oral↗

PubMed2004

[Analysis of computed tomography-based distribution of metastatic cervical nodes in 218 cases of nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: The application of intensity- modulated radiation therapy (IMRT) for nasopharyngeal carcinoma (NPC) requires a precise delineation of the nodal area and nodal clinical target volume (CTV) on computed tomography (CT) images,and the prerequisite is to find out the rules of CT-based distribution of metastatic lymph nodes of NPC. This study was designed to analyze the rules of CT-based distribution of nodal involvements of NPC according to the guidelines of nodal levels proposed by Radiation Therapy Oncology Group (RTOG). METHODS: From Jul. 2003 to Nov.2003, 259 newly diagnosed NPC patients received radiotherapy at Fudan University Affiliated Cancer Hospital. All patients had transversal contrast enhanced CT scan from base of skull to clavicle before treatment. Diagnostic radiologists and radiation oncologists together assessed the nodal distribution in each RTOG nodal level. Chi-square test was used to analyze the correlation between T stage and nodal metastasis rate. The neck was further divided into 3 regions by the verge of hyoid bone and the inferior border of cricoid cartilage to assess leap metastasis of nodes. RESULTS: A total of 218 patients (84.2%)had nodal involvement. The distribution was as follow: 0 in level Ia, 6 (2.8%) in level Ib, 115 (52.8%) in level IIa,192 (88.1%) in level IIb, 78 (35.8%) in level III, 20 (9.2%) in level IV, 65 (29.9%) in level V, 0 in level VI,157 (72.0%)in retropharynx, and 2 (0.9%) at preauricular area. Leap metastases were found in only 5 patients (2.3%). No significant correlation was found between T stage and nodal involvement. CONCLUSION: NPC has a high probability of nodal metastases, nodes in level IIa,IIb, and retropharynx are most likely to be involved. Nodes metastasized mostly from the upper to the lower level, and from the proximal to the distal part, with a very low leap metastasis rate. The relationship between T stage and nodal involvement has no statistical significance.

Adolescent↗

PubMed2004

Significant associations of the alpha-adducin gene Gly460Trp polymorphism with serum bilirubin concentrations in Chinese essential hypertension patients.

We investigated associations of the Gly460Trp polymorphism of the alpha-adducin gene and concentrations of serum total bilirubin, serum direct bilirubin and serum unconjugated bilirubin in patients with essential hypertension from Anhui, China from September 2000 to January 2001. Compared to women with the Gly/Gly genotype and after adjustment for important covariates, women with the Trp/Trp genotype had lower mean concentrations of serum total bilirubin (beta = -1.2 micromol/L; P = 0.01), serum direct bilirubin (beta = - 0.4 micromol/L; P = 0.02) and serum unconjugated bilirubin (P = -0.8 micromol/L; P = 0.03). Among women in either the upper or lower quartiles of serum total bilirubin, serum direct bilirubin and serum unconjugated bilirubin and compared to those with the Gly/Gly genotype, women with the Trp/Trp genotype had higher odds of being in the lower quartile of concentrations of serum total bilirubin (odds ratio = 4.0; 95 percent confidence interval: 1.6 - 10.2; P < 0.01), serum direct bilirubin (odds ratio = 4.0; 95 percent confidence: 1.6 - 9.7; P < 0.01) and serum unconjugated bilirubin (odds ratio = 2.7; 95 percent confidence interval: 1.1 - 6.7; P = 0.03) after adjustment for important covariates. We did not observe any significant associations in these models for men. We concluded that the Trp/Trp genotype of alpha-adducin Gly460Trp was associated with lower serum bilirubin concentrations in this group of Chinese women with essential hypertension. Women with the Trp/Trp genotype of alpha-adducin Gly460Trp might have increased risk for cardiovascular diseases due to lower concentrations of serum bilirubin.

Adult↗

PubMed2004

Helicobacter bilis-associated hepatitis in outbred mice.

Although Helicobacter bilis infects mice worldwide, it is not known whether H. bilis causes enterohepatic disease in outbred Swiss Webster (SW) mice. Intestinal and liver specimens from four groups of 39 SW mice, five of which were treated with creatine in the drinking water, were obtained for culture for the presence of H. bilis and were analyzed as to whether infection status was associated with H. bilis seroconversion and/or hepatitis. Helicobacter bilis was isolated from the colon of all 27 mice of groups I-III, but only from the liver of one 12- to 13-month-old female mouse. Ten of 27 livers were H. bilis-positive based on polymerase chain reaction (PCR) analysis; 8 of 10 (80%) of the positive results were for older mice. Results of PCR analysis for H. bilis were negative, and H. bilis was not isolated from 12 control mice (group IV). Irrespective of treatment group or controls, severity of histologic lobular and periportal chronic inflammatory lesions in the liver of H. bilis-infected outbred mice ranged from minimally to moderately severe. Helicobacter bilis infection was associated with increased portal inflammation in group III mice, compared with age-matched, helicobacter-free, group IV mice (P < 0.03). A comparison of potential sex effects in group III mice indicated that H. bilis-infected female mice developed more severe portal inflammation than did H. bilis-infected male mice (P < 0.01). On the basis of results of an ELISA, 8 of 11, 6- to 8-month-old H. bilis-infected mice of group III seroconverted to H. bilis outer membrane antigen. Helicobacter bilis infection is associated with hepatitis in SW mice and can confound experimental results.

Administration, Oral↗

PubMed2003

Association between polymorphism of the dopamine transporter gene and early smoking onset: an interaction risk on nicotine dependence.

Previous studies suggested that a polymorphism in the dopamine transporter gene (SLC6A3) is associated with nicotine dependence and age of smoking onset, but the conclusion was controversial. To detect the association of a G-->A polymorphism (NCBI dbSNP cluster ID: rs27072) in 3'-untranslated region of the SLC6A3 with nicotine dependence and early smoking onset, we recruited 253 sibships including 668 nicotine-dependent siblings from a rural district of China. The sibship disequilibrium tests (SDT) showed that the rs27072-A allele is significantly associated with smoking onset < or =18 years (kappa2=9.78, p=0.003 in severely nicotine-dependent smokers, and kappa2=4.24, p=0.058 in total smokers), but not significantly associated with severe nicotine dependence. Conditional logistic regression showed that the risk of early smoking onset by the rs27072-A allele was almost three times greater in severely nicotine-dependent smokers [Odds ratio (OR)=11.3, 95% confidence interval (CI)=1.5-85.6] than that in total smokers. Linear regression showed that rs27072-A allele also increased the risk of nicotine dependence by early smoking onset compared with homozygous rs27072-G genotype. Although these findings are preliminary and need validation, the results suggest that a polymorphism in the SLC6A3 may play important roles in smoking onset, and there may be an interactive effect between the SLC6A3 and early smoking onset on modulating the susceptibility of nicotine dependence.

Age of Onset↗

PubMed2003

Isolation and characterization of novel Helicobacter spp. from the gastric mucosa of harp seals Phoca groenlandica.

Since the recent discovery of Helicobacter cetorum in cetaceans and its role in the development of gastritis, speculation has existed as to whether pinnipeds have Helicobacter spp. associated gastritis and peptic ulcer disease. The gastric mucosa of 4 stranded harp seals Phoca groenlandica from the Massachusetts coastline were assessed for Helicobacter spp. by culture and PCR. We cultured 2 novel Helicobacter spp. from the pyloric antrum of 1 of the 4 harp seals studied, and identified these by PCR in 2 of the 4 seals. Both gram-negative bacterial isolates were catalase- and oxidase-positive. However, a fusiform helicobacter with flexispira morphology was urease-positive, and a spiral-shaped helicobacter was urease-negative. Slender, spiral and fusiform-shaped bacteria were detected in the gastric mucosa by the Warthin-Starry stain. Histopathologic analysis revealed mild diffuse lymphoplasmacytic gastritis within the superficial mucosa of the pyloric antrum of both infected seals. The 2 bacterial isolates were classified by 16S rRNA analysis; they clustered with other enteric helicobacters and represent 2 novel Helicobacter spp. The urease-negative bacterial isolate clustered with H. canis and the urease-positive isolate clustered with an isolate from a sea lion and isolates from sea otters. This cluster of pinniped isolates has 97 % similarity to a number of Helicobacter species, but appears to be most closely related to other helicobacters with flexispira morphology. These findings suggest that the novel Helicobacter spp. may play a role in the etiopathogenesis of gastrointestinal diseases in pinnipeds. To our knowledge, this represents the first isolation and characterization of a novel Helicobacter spp. from pinnipeds.

Animals↗

PubMed2003

CD4(+)CD25(+) regulatory lymphocytes require interleukin 10 to interrupt colon carcinogenesis in mice.

Roles for host immune response in carcinogenesis are not well defined. Recent studies have shown that microbially driven inflammation can lead to colon cancer and that prior transfer of regulatory lymphocytes expressing CD4 and CD25 prevents the innate inflammatory events that lead to colon cancer in mice. To further examine the ability of regulatory lymphocytes to inhibit carcinogenesis, 129/SvEv Rag-2-deficient mice were inoculated by gastric gavage with Helicobacter hepaticus, an enteric bacterial pathogen of mice. Mice were then treated at 1, 3, or 12 months after infection with adoptive transfer of CD4(+)CD45RB(lo)CD25(+)-regulatory cells. Mice dosed with regulatory cells at 4 or 12 weeks after H. hepaticus infection had reduced severity of inflammatory bowel disease and significantly lower risk of colon cancer during the 8 month observation period, compared with infected mice that had not received cells. This suggested that regulatory cells were able to interrupt the ongoing innate immune events in the stepwise progression to cancer. Transfer of regulatory cells into chronically infected mice with established cancer reduced severity of colitis, epithelial dysplasia, and cancer, but did not eliminate all tumors. Regulatory cells lacking anti-inflammatory cytokine interleukin (IL)-10 were unable to inhibit inflammatory bowel disease, dysplasia, or cancer, showing that IL-10 was required for the protective effects of lymphocytes in this setting. Taken together, the data suggest that IL-10-mediated suppression of host innate inflammatory response was pivotal in interrupting carcinogenesis. Regulatory lymphocytes and cytokines may have implications for novel therapies for colon cancer in humans.

Adenocarcinoma↗

PubMed2003

Exo1: a new chemical inhibitor of the exocytic pathway.

A phenotypic screen was used to search for drug-like molecules that can interfere with specific steps in membrane traffic. 2-(4-Fluorobenzoylamino)-benzoic acid methyl ester (Exo1), identified in this screen, induces a rapid collapse of the Golgi to the endoplasmic reticulum, thus acutely inhibiting the traffic emanating from the endoplasmic reticulum. Like Brefeldin A (BFA), Exo1 induces the rapid release of ADP-ribosylation factor (ARF) 1 from Golgi membranes but has less effect on the organization of the trans-Golgi network. Our data indicate that Exo1 acts by a different mechanism from BFA. Unlike BFA, Exo1 does not induce the ADP-ribosylation of CtBP/Bars50 and does not interfere with the activity of guanine nucleotide exchange factors specific for Golgi-based ARFs. Thus, Exo1 allows the fatty acid exchange activity of Bars50 to be distinguished from ARF1 activity in the control of Golgi tubulation.

Adenosine Diphosphate Ribose↗

PubMed2003

Determination of Simvastatin in human plasma by liquid chromatography-mass spectrometry.

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for the determination of simvastatin (I) has been developed. After extraction by ethyl acetate, using lovastatin (II) as internal standard, solutes are separated on a C(18) column with a mobile phase consisting of methanol-water (9:1). Detection is performed on an atmospheric pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operates in positive ionization mode. Simvastatin quantification was realized by computing peak area ratio (I/II) of the extracts analyzed in SIM mode (m/z: 441 and m/z: 427 for I and II, respectively) and comparing them with calibration curve (r=0.9997). Accuracy and precision for the assay were determined by calculating the intra-batch and inter-batch variation at three concentrations 0.1, 5.0, 10.0 ng/ml; the intra batch relative standard deviation (RSD) was less than 10% and ranged from 1.8 to 8.5%, respectively; the inter-batch RSD was less than 20% and ranged from 4.1 to 16.5%. The limit of detection was 0.05 ng/ml.

Adult↗

PubMed2003

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii.

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.

Amino Acid Sequence↗

PubMed2003

Structure based hyperthermostability of archaeal histone HPhA from Pyrococcus horikoshii.

The histone protein HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii, shows hyperthermostability, as required for optimal growth of the organism at 95 degrees C. The structure of recombinant P.horikoshii HPhA has been determined to 2.3A resolution by molecular replacement, and refined to R(work) and R(free) values of 20.7% and 27.3%, respectively. The HPhA monomer structure is characterized by the histone fold and assembles into a homodimer like other archaeal histones. There are four anions found in the dimer structure, giving rise to clues as to where DNA might bind. A detailed comparison of four known structures of archaeal histones, which evolve and exist under different temperatures, shows that the thermophilic archaeal histone HPhA has a larger hydrophobic contact area, an increased number of hydrogen bonds and a reduced solvent-accessible area. We also observe a unique network of tyrosine residues located at the crossover point of the two HPhA monomers, which locks them together and stabilizes the dimer. These factors together account for the increased thermal stability.

Amino Acid Sequence↗

PubMed2003

Human VPS34 and p150 are Rab7 interacting partners.

Regulation of membrane trafficking requires the concerted actions of rab proteins, their effectors and several phosphatidylinositol 3'-kinases. Rab7 is required for late endosomal transport and here we establish that the phosphatidylinositol 3'-kinase hVPS34 and its adaptor protein p150 are rab7 interacting partners. The hVPS34/p150 complex colocalized with rab7 on late endosomes and hVPS34 activity was dependent on nucleotide cycling of rab7. In addition, total cellular phosphatidylinositol 3'-phosphate levels were modulated by rab7 expression, suggesting that rab7 activation impacted kinase cycling to early endosomes. The data identify rab7 as an important regulator of late endosomal hVPS34 function and link rab7 to the regulation of phosphatidylinositol 3'-kinase cycling between early and late endosomes.

Animals↗