Biomedical subjects

Mei Hu

Publications and source records attributed to Mei Hu.

10 recordsLinked to original sources

PubMed2005

An immunosensor for ferritin based on agarose hydrogel.

A novel electrochemical immunosensor for determination of ferritin in serum has been proposed. The immunosensor was prepared by immobilizing ferritin antibody (FeAb) on a glassy carbon electrode (GCE) based on agarose hydrogel. The modification procedure of the immunosensor was characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The effects of amount of FeAb, incubation time and temperature on the immunosensor were explored to provide optimum analytical performance. The determination of ferritin was based on the change in DPV response before and after the antibody (Ab)-antigen (Ag) reaction. Tests result indicated that FeAb in the device microenvironment had biological activity. The detection limit for ferritin was 1.5 x 10(-5) g l(-1) and the linear range was 5-50 x 10(-5) g l(-1) with a correlation coefficient of 0.996. The storage stability was acceptable in pH 7.0 phosphate buffer saline (PBS) solution at 4 degrees C for 10 days. The proposed immunosensor provides a new promising method for the clinical immunoassay of ferritin.

Antibodies↗

PubMed2005

The penC mutation conferring antibiotic resistance in Neisseria gonorrhoeae arises from a mutation in the PilQ secretin that interferes with multimer stability.

The penC resistance gene was previously characterized in an FA19 penA mtrR penB gonococcal strain (PR100) as a spontaneous mutation that increased resistance to penicillin and tetracycline. We show here that antibiotic resistance mediated by penC is the result of a Glu-666 to Lys missense mutation in the pilQ gene that interferes with the formation of the SDS-resistant high-molecular-mass PilQ secretin complex, disrupts piliation and decreases transformation frequency by 50-fold. Deletion of pilQ in PR100 confers the same level of antibiotic resistance as the penC mutation, but increased resistance was observed only in strains containing the mtrR and penB resistance determinants. Site-saturation mutagenesis of Glu-666 revealed that only acidic or amidated amino acids at this position preserved PilQ function. Consistent with early studies suggesting the importance of cysteine residues for stability of the PilQ multimer, mutation of either of the two cysteine residues in FA19 PilQ led to a similar phenotype as penC: increased antibiotic resistance, loss of piliation, intermediate levels of transformation competence and absence of SDS-resistant PilQ oligomers. These data show that a functional secretin complex can enhance the entry of antibiotics into the cell and suggest that the PilQ oligomer forms a pore in the outer membrane through which antibiotics diffuse into the periplasm.

Amino Acid Substitution↗

PubMed2005

High-level chromosomally mediated tetracycline resistance in Neisseria gonorrhoeae results from a point mutation in the rpsJ gene encoding ribosomal protein S10 in combination with the mtrR and penB resistance determinants.

Neisseria gonorrhoeae becomes resistant to tetracycline by two major mechanisms: expression of a plasmid-encoded TetM protein and mutations in endogenous genes (chromosomally mediated resistance). Early studies by Sparling and colleagues (P. F. Sparling F. A. J. Sarubbi, and E. Blackman, J. Bacteriol. 124:740-749, 1975) demonstrated that three genes were involved in high-level chromosomally mediated tetracycline resistance (MIC of tetracycline > or = 2 microg/ml): ery-2 (now referred to as mtrR), penB, and tet-2. While the identities of the first two genes are known, the tet-2 gene has not been identified. We cloned the tet-2 gene, which confers tetracycline resistance, from tetracycline-resistant clinical isolate N. gonorrhoeae FA6140 and show that resistance is due to a single point mutation (Val-57 to Met) in the rpsJ gene (rpsJ1) encoding ribosomal protein S10. Moreover, the identical mutation was found in six distinct tetracycline-resistant clinical isolates in which the MIC of tetracycline was > or =2 microg/ml. Site-saturation mutagenesis of the codon for Val-57 identified two other amino acids (Leu and Gln) that conferred identical levels of resistance as the Met-57 mutation. The mutation maps to the vertex of a loop in S10 that is near the aminoacyl-tRNA site in the structure of the 30S ribosomal subunit from Thermus thermophilus, and the residue equivalent to Val-57 in T. thermophilus S10, Lys-55, is within 8 to 9 A of bound tetracycline. These data suggest that large noncharged amino acids alter the rRNA structure near the tetracycline-binding site, leading to a lower affinity of the antibiotic.

Alleles↗

PubMed2005

[Serological antibodies comparison of a hepatitis E outbreak].

OBJECTIVE: To compare the serological characters of an outbreak of hepatitis E and evaluate sensitivity and specificity of anti-HEV E2-IgM. METHODS: The sera collected from the employees of an outbreak unit were detected for anti-HEV E2-IgM and IgG, and the serum samples from a neighboring department were used as control. The results detected with anti-HEV E2-IgM, IgG and Genelab anti-HEV IgM, IgG in some samples were compared. RESULTS: The positive rate of anti-HEV E2-IgM in the control group was 0.11%. The results between the positive and the negative samples can be distinguished easily. The specificity of anti-HEV E2-IgM is about 99.89%. The positive rate of anti-HEV E2-IgM in outbreak stricken population was 8.66%, significantly higher than that in the control group (P < 0.001). The results from HEV patients' serial samples in the outbreak unit showed that the anti-HEV E2-IgM titer was high 30-60 days after the infected and then declined clearly. The positivity seemed unrelated to neither sex nor age. Among the 115 positive to anti-HEV E2-IgM, 27 were negative to Genelab anti-HEV IgG, the fact indicated a rather high risk of misdiagnosis of about 23.48%. In the 179 randomized samples of the control group, the positive rate of Genelabs anti-HEV IgG was about 11.17%. In 110 samples for the positive anti-HEV E2-IgM, the positive ratio of Genelabs anti-HEV IgG was about 76.36%, and that of Genelabs anti-HEV IgM only 69.09%. There were 16 samples negative for both Genelabs anti-HEV IgG and IgM. The ratio of the difference between the Genelabs anti-HEV IgG and IgM was about 25.45%. CONCLUSION: The specificity of anti-HEV E2-IgM was about 99%, and false positive rate was low. The sensitivity of anti-HEV E2-IgM in acute hepatitis E infection was 25%-30% higher than that of Genelabs anti-HEV IgM,IgG. The infected persons in the outbreak unit can be preferably distinguished from the non-infected persons by anti-HEV E2-IgM. Anti-HEV E2-IgM can image the characters of the outbreak of HEV and played a great role in the control of outbreak and in the early diagnosis for hepatitis E.

Antibodies, Viral↗

PubMed2004

Integrated high capacity solid phase extraction-MS/MS system for pharmaceutical profiling in drug discovery.

A method is described for use in analysis of samples from pharmaceutical profiling of early drug discovery compounds. The method consists of a high capacity autosampler which injects samples into one of two solid phase extraction columns operated in parallel for alternating trapping, washing and elution into a tandem quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) MS/MS mode. A primary method, which is useful for 80-90% of compounds, and a secondary method, which is useful for a majority of the remaining compounds, are described. No analytical HPLC column is used and the analysis rate is approximately 50 samples/h. Specificity is obtained using MRM analysis. Application of the method for high capacity analysis of metabolic stability samples is described.

Drug Design↗

PubMed2003

Interconversion of ATP binding and conformational free energies by tryptophanyl-tRNA synthetase: structures of ATP bound to open and closed, pre-transition-state conformations.

Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at approximately 8mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the [ATP] over the same concentration range ( approximately 5mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1mM ATP and a low-affinity complex grown at 10mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7A. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2A) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the alpha and gamma-phosphates; interactions of the K111 side-chain with the gamma-phosphate; and a water molecule bridging the consensus sequence residue T15 to the beta-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP(i) group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high [ATP] implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.

Adenosine Triphosphate↗

PubMed2003

[Serological characteristics of a hepatitis E outbreak].

OBJECTIVE: To look into the serological characteristics of a hepatitis E outbreak. METHODS: Sera from the first five patients with acute icteric hepatitis who developed the disease successively within ten days and the 1,675 employees routinely having their lunch in a dining hall of a department (outbreak population) were examined for anti.HEV IgM and IgG at 26th days after the outbreak, and the 883 employees of a neighboring department not having their lunch in the hall were selected as control (control population). RESULTS: The five patients were all positive for anti-HEV IgM and IgG. The positive rates of anti-HEV IgM and IgG in outbreak population were 8.7% and 38.4% respectively, both significantly higher than those in control population which were only 0.1% and 28.6%. The numbers with abnormal ALT in the 145 individuals with anti-HEV IgM(+) of outbreak population were significantly higher than those in the IgM(-) individuals of the same group as well as in control, while the abnormal ALT ratio in the IgM(-) individuals of the outbreak was not higher than that in control. The results from the four patients' serial sera showed that the anti-HEV IgM titers declined gradually and were undetectable at about 4th month after infection, and the IgG titers increased to peak in about 2-3 months after infection, then declined very slowly. The mean IgG titer of the anti-HEV IgM(+) individuals was significantly higher than that of the IgM(-) but IgG(+) individuals in outbreak population, and the latter was significantly higher than the IgG(+) individuals in control, which suggested that the post-infection individuals' immunities to HEV were boosted during the outbreak. There was no difference between sex or age groups for the anti-HEV IgM(+) ratio, but the abnormal ALT was much more frequent in the anti-HEV IgM(+) male than in the female, and no difference was observed between age groups. CONCLUSION: The pathogen of the outbreak of acute icteric hepatitis was hepatitis E virus and associated with food intake. Anti-HEV IgM and IgG were used not only for diagnosis of hepatitis E but also for surveilance in mass population. The attack risk was not associated with age or sex, but the abnormal ALT was much more frequent fresh infectors in male.

Adult↗

PubMed2002

Characterization of 11 human sarcoma cell strains: evaluation of cytogenetics, tumorigenicity, metastasis, and production of angiogenic factors.

BACKGROUND: Human sarcomas have a propensity for aggressive local invasion and early pulmonary metastasis. Frequently, deaths are due to uncontrolled pulmonary metastases. The purpose of the current study was to evaluate cytogenetics, tumorigenicity, metastatic potential, and production of angiogenic factors in human sarcoma cell strains. A secondary purpose was to establish low passage cell strains for studying new therapeutic approaches. METHODS: The authors established 11 cell strains from human sarcoma surgical specimens and characterized their in vitro tumor properties, including growth in soft agar, expression of angiogenic growth factors (vascular endothelial growth factor [VEGF] and basic-fibroblast growth factor [bFGF]), and cytogenetics. RESULTS: All of the cell strains remained diploid. All exhibited the ability to grow in soft agar and expressed both VEGF as well as bFGF. In addition, 6 of the 11 established sarcoma cell strains were tumorigenic, 5 of which spontaneously metastasized to the lungs in nude mice. Four of the five cell strains that yielded lung metastases were derived from lung metastases in patients. CONCLUSIONS: The 11 cell strains, which were derived from diverse sarcoma histologies, will provide a model for studying not only metastatic progression but also the in vitro and in vivo efficacy of new therapeutic modalities for human sarcomas.

Animals↗

PubMed2002

Nicotinic regulation of CREB activation in hippocampal neurons by glutamatergic and nonglutamatergic pathways.

Activity-dependent gene expression is essential for form and function in the nervous system. Best understood is the role of glutamatergic signaling in controlling such events, but nicotinic signaling can also regulate transcription. We show here that nicotine can alter gene expression in rat hippocampal neurons, as reflected by activation of the transcription factor CREB and appearance of the immediate early gene product c-Fos. The process depends on both CaM and MAP kinases and on calcium release from internal stores. Part of the nicotinic effect is mediated via glutamatergic transmission, even in the absence of action potentials. Voltage-gated calcium channels are not necessary for nicotine-induced activation of CREB in hippocampal neurons. The low levels of sustained nicotinic stimulation required for transcriptional effects are consistent with those likely to be achievable either by the normal septal cholinergic innervation of the hippocampus or by repeated tobacco usage.

Acetylcholine↗

PubMed2002

Mutations in ponA, the gene encoding penicillin-binding protein 1, and a novel locus, penC, are required for high-level chromosomally mediated penicillin resistance in Neisseria gonorrhoeae.

Chromosomally mediated penicillin resistance in Neisseria gonorrhoeae occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of N. gonorrhoeae to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains for which MICs of penicillin were > or = 1 microg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation (k2/K') than wild-type PBP 1 with a variety of beta-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered ponA gene (ponA1) in several CMRNG strains with the wild-type ponA gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5) with the ponA1 gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed penC, which was required along with ponA1 to increase penicillin resistance of PR100 to a high level (MIC = 4 microg/ml). The penC locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus, penC, is required along with ponA1 to achieve high-level penicillin resistance.

Anti-Bacterial Agents↗