Crystal structure of the HhaI DNA methyltransferase.
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Amino Acid Sequence↗
Biomedical subjects
X Cheng
Publications and source records attributed to X Cheng.
Effects of antibody isotype and host cell type on in vitro neutralization of Chlamydia trachomatis.
Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapping peptides in the variable domain IV of the major outer membrane protein of Chlamydia trachomatis but differ in isotype, were used in a complement-independent (CI) in vitro neutralization assay. These MAbs had previously been shown to neutralize chlamydial infectivity in HeLa 229 cells in a complement-dependent assay. In this report, all three MAbs neutralized chlamydial infectivity in HaK cells in a CI assay. However, when HeLa cells were used as the host cell, MAb E-4 (immunoglobulin G2b [IgG2b]) and MAb DIII A3 (IgG2b) failed to neutralize infectivity, while MAb E-21 (IgG1) neutralized chlamydial infectivity. These findings are consistent with the proposal that because of the presence of Fc gamma RIII receptors, HeLa cells facilitate infectivity and thus block neutralization through the uptake of an IgG2b-chlamydia complex. Since Fc gamma RIII receptors do not bind or bind poorly to IgG1, neutralization of C. trachomatis by MAb E-21 in HeLa cells is also corroborative evidence for the role of Fc gamma RIII receptors in this interaction. A fivefold enhancement of infectivity was seen when 10 and 1 micrograms of MAb E-4 per ml were tested in a CI assay with HeLa cells. In performing CI neutralization synergy studies in HeLa cells with MAbs E-4 and E-21, antagonism between MAbs E-4 and E-21 was observed at MAb E-4 concentrations of 10 and 1 micrograms/ml for all concentrations of MAb E-21 tested (10 to 0.1 micrograms/ml). When HaK cells were used in the same studies, no antagonism between the MAbs was found. In addition, when HeLa cells were used in a CI assay, polyclonal serum raised to a peptide representing variable domain IV of the major outer membrane protein inhibited the neutralizing ability of MAb E-21. The blocking of neutralization and the enhancement of infectivity by chlamydia-specific antibodies seen in this investigation with HeLa cells may have important clinical implications for developing preventive strategies for chlamydial infections.
Amino Acid Sequence↗
Characterization of a neutralizing monoclonal antibody directed at variable domain I of the major outer membrane protein of Chlamydia trachomatis C-complex serovars.
A monoclonal antibody (MAb), C10, that neutralized in vitro the infectivity of serovars C, I, J, and L3 (members of the C and C-related complexes) of Chlamydia trachomatis was identified. Of the 15 major serovars and the mouse pneumonitis strain of C. trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae, which were used as nontreated and heat-treated (56 degrees C, 30 min) antigens in a dot blot assay, only serovars C, I, J, and L3 were recognized with both the native and treated antigens. Western blot (immunoblot) results showed that MAb C10 recognized the major outer membrane protein of these four serovars. Overlapping hexameric peptides corresponding to variable domains (VDs) I, II, III, and IV of the major outer membrane protein of C. trachomatis serovar C were synthesized, and peptide screening showed that MAb C10 mapped to the VD I amino acid sequence VAGLQNDPT. Results of an in vitro neutralization assay correlated with those of the indirect immunofluorescence assay, Western blot, and dot blot assay in that only serovars C, I, J, and L3 were neutralized by MAb C10. In vitro competitive neutralization experiments, using a peptide representing VD I of serovar C to compete with C. trachomatis serovar C for MAb C10 binding, revealed that both serological and neutralizing activities of MAb C10 were inhibited by the VD I peptide. In an in vivo toxicity/infectivity assay using serovar L3 pretreated with MAb C10, there was 100% survival of mice infected with a lethal dose at 48 h. In contrast, the control group, consisting of mice injected with the same dose of L3 pretreated with a MAb that does not recognize L3, had no survivors during a 48-h observation period. In summary, since the surface-exposed contiguous epitope recognized by MAb C10 binds neutralizing antibodies that are subspecies specific for the C and C-related complexes, it should be considered for inclusion in the development of a chlamydial vaccine.
Amino Acid Sequence↗
Identification and characterization of serotype 4-specific antigens of Ureaplasma urealyticum by use of monoclonal antibodies.
Monoclonal antibodies against Ureaplasma urealyticum serotype 4 were produced by immunizing BALB/c mice with whole-cell antigens of the U. urealyticum serotype 4 reference strain. Ten monoclonal antibodies differentiated into two groups were found: one group included five monoclonal antibodies recognizing a band in immunoblotting that had a molecular mass of 81 kDa, and a second group included another five monoclonal antibodies recognizing three bands in immunoblotting that had molecular masses of 81, 75, and 71 kDa. Fifteen clinical U. urealyticum isolates were selected for serotyping with serotype 4-specific monoclonal antibodies and polyclonal antisera 1 to 14. The results obtained with polyclonal and monoclonal antibodies suggest the existence of heterogeneity of the serotype antigens among clinical isolates of U. urealyticum serotype 4.
Antibodies, Bacterial↗
Combination of traditional Chinese and Western medicine in treating acute posterior multifocal placoid pigment epitheliopathy.
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Adolescent↗
[Ecogeographical distribution, utilization and protection of rare and precious traditional Chinese drug Changium smyrnioides Wolff].
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Conservation of Natural Resources↗
Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex.
The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.
Crystallization↗
Construction and use of lambda PL promoter vectors for direct cloning and high level expression of PCR amplified DNA coding sequences.
A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed. The vectors contain the phage lambda PL promoter, a synthetic translation initiation region (TIR), and convenient cloning sites. The cloning sites provide all or part of an AUG translation initiation codon and facilitate the precise fusion of target DNA sequences to vector transcriptional and translational signals. The vectors were constructed with synthetic TIRs because there is evidence which suggests that the efficiency of the phage lambda cII gene TIR present in the parental vector depends strongly on information contained within the cII N-terminal coding sequence. Bovine brain 14-3-3 eta chain cDNA was PCR-amplified and used to demonstrate the expression capacity of the newly constructed vectors. A significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR. Expression levels vary from 15% to 48% of total cell protein. The effects of a reported translational enhancer from phage T7 on expression of the 14-3-3 protein are also discussed. The vectors should be generally useful for high level heterologous protein expression in Escherichia coli.
Bacteriophage lambda↗
Metabolism of trans,trans-muconaldehyde by aldehyde and alcohol dehydrogenases: identification of a novel metabolite.
The metabolism of trans,trans-muconaldehyde (MA), a highly reactive alpha,beta-unsaturated dialdehyde, was examined in vitro using purified yeast alcohol and aldehyde dehydrogenases (ADH and ALDH, respectively). In the presence of NAD(+)-fortified ALDH, the mono-oxidation product (acid/aldehyde) was the primary metabolite formed with trace amounts of the dioxidation product (trans,trans-muconic acid). In NADH-fortified reactions with ADH, both the mono- and direduction products (hydroxy/aldehyde and dihydroxy, respectively) were readily detected. Oxidation and reduction products of MA were formed in incubates containing both dehydrogenases together with either NAD+ or NADH. Unexpectedly, an additional metabolite was detected, which was a major product in both NAD(+)- and NADH-fortified systems containing ALDH and ADH in combination and whose formation could be inhibited by pyrazole (an ADH inhibitor). ALDH-mediated oxidation of a synthetic standard of the hydroxy/aldehyde derivative of MA resulted in formation of this new metabolite, which was also a major product formed by rat hepatocytes incubated with MA. Using HPLC/photodiode array detection, the new metabolite was found to cochromatograph and have a uv spectrum identical to that of a synthetic standard of the hydroxy/acid derivative of MA. The metabolite was confirmed as the hydroxy/acid derivative of MA after preparative HPLC, TMS derivatization, and GC/MS analysis. The hydroxy/acid metabolite was not formed during ADH-mediated reduction of the mono-oxidation product of MA, suggesting that this metabolite was formed by yeast dehydrogenases via a primary reduction of MA and subsequent oxidation of the hydroxy/aldehyde to the hydroxy/acid. These data show that the hydroxy/acid derivative is a novel metabolite of MA, which arises from the interaction of both oxidative and reductive routes of metabolism.
Alcohol Dehydrogenase↗
Characterization of the humoral response induced by a peptide corresponding to variable domain IV of the major outer membrane protein of Chlamydia trachomatis serovar E.
A 30-amino-acid peptide corresponding to variable domain IV (VD IV) of the major outer membrane protein of Chlamydia trachomatis serovar E was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize mice. The resulting antisera (anti-KLH-VD IV sera) recognized all 15 serovars of C. trachomatis when assayed by indirect immunofluorescence and Western blotting. Probing of overlapping hexameric peptides representing VD IV with mouse anti-KLH-VD IV sera revealed that two main regions of the peptide were recognized by the antisera, the N terminus of the peptide, which contains B-complex-specific epitopes, and the middle region of the peptide, which contains a species-conserved domain. When used in an in vitro neutralization assay, these antisera were able to neutralize mainly serovars in the B complex. These data provide evidence that a linear peptide corresponding to VD IV can induce in vitro protection from C. trachomatis infectivity that is subspecies specific.
Animals↗
[Conditions for dextranase formation by Paecilomyces lilacinus].
Induced formation conditions of dextranase by Paecilomyces lilacinus were investigated. Effect of various carbohydrates on dextranase formation was examined, dextran was the best C-source and as an inducer. The effect of dextran with different molecular weight (from 17.2 to 1000 kD) on dextranase formation was compared, productivity of dextranase increased with increase of dextran molecular weight. When dextran of 1000 kD was used as C-source. The enzyme formation was 40% higher than that 17.2 kD dextran. When other sugars were separately added to the medium with dextran, the enzyme formation was repressed. Besides C-source, the other optimum conditions of dextranase formation were as follows: N-source, beef peptone; medium initial pH, 6.0-7.0; culture temperature, 28 degrees C; inoculum amount about 10%, and the organism was cultivated for 6 days on 200 r/min shaker in 250 ml flasker with 50 ml medium.
Carbohydrates↗
[Effect of circadian rhythm on the action of acupuncture to prevent the formation of experimental gastric ulcer].
Taking rats and mice as subjects, observation had been made on the protecting action of acupuncture to prevent the gastric mucosa from experimental injury. The pH value (by accurate test paper), gastric acid output (by titration), the secretion of PG in gastric mucosa (by radioimmunoassay) were observed altogether at 1-3h, 5-7h, 9-11h, 13-15h, 17-19h and 21-23h. All of the above indices were changed along with the alternation of day and night. And the effects of acupuncture on these indices were different when giving acupuncture at different times. The action of acupuncture to prevent experimental gastric ulcer was also different at different times. It is the general regularity that puncturing at the acrophase of circadian rhythm, the effect was mainly inhibitory, while puncturing at the valleyphase, the effect was mostly excitatory. Therefore, utilizing the influence of the various phases of circadian rhythm on the acupuncture effect, to choose the optimum time giving acupuncture could be one way to promote the acupuncture curative effect.
Acupuncture Therapy↗
[Analysis of natural history and long-term follow-up of 59 patients with obliterative pulmonary hypertension (OPH)].
The diagnosis of OPH was made by clinical and hemodynamic data in 59 patients, including 30 cases of unexplained pulmonary hypertension, 26 cases of pulmonary embolism and 3 others. The survival curve showed that the two-year survival rate of the patients was 76.72%, and the five-year rate was 63.37%. The single correlation factors influencing prognosis were the interval from onset of symptoms to diagnosis (course), cardiac function, PaO2, SaO2, cause of disease, pulmonary artery pressure and right atrial pressure. The outcome of long-term treatment (mean 4.2 years) in 15 patients with OPH was as follows: remarkable improvement in 8 cases, amelioration in 3, no change in 2 and deterioration in 2. The total effective rate was 73.33%. The importance of anticoagulant therapy and the use of nifedipine are discussed.
Adolescent↗
Functional and structural mapping of Chlamydia trachomatis species-specific major outer membrane protein epitopes by use of neutralizing monoclonal antibodies.
Three monoclonal antibodies (MAbs), E4, L1-4, and L1-24, to the major outer membrane protein (MOMP) of Chlamydia trachomatis were identified that neutralized in vitro the infectivity of members of the B- and C-related complex as well as the mouse pneumonitis strain. MAbs L1-4, L1-24, and E4 gave a strong signal in an indirect immunofluorescence assay and/or Western immunoblot with all serovars of the lymphogranuloma venereum and trachoma biovars and a weak signal with the mouse biovar. In addition, C. psittaci and C. pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by the technique of pneumoniae were also weakly recognized by MAbs L1-4 and L1-24. As determined by by the technique of overlapping peptides, all three MAbs showed reactivity to variable domain (VD) IV of MOMP. While all three MAbs had different recognition patterns, all strongly bound to the peptides TLNPTI and LNPTIA within the species-conserved region of VD IV. MAb E4 also recognized the peptide SATAIF in the subspecies region of VD IV. Peptides corresponding to VD IV of MOMP were synthesized and used in competitive inhibition experiments to determine the functional location of the epitope recognized by these three MAbs. Both the serological and neutralizing activities of MAb E4 were inhibited by the peptides ATAIFDTTTLNPTIAG and FDTTTLNPTIAG; however, none of the peptides made to the VD IV region blocked the neutralizing activity of MAbs L1-4 and L1-24. Therefore, the neutralizable domain of the epitope recognized by MAb E4 is contiguous and may be an important candidate for inclusion in a subunit vaccine.
Amino Acid Sequence↗
[Androgen-like and anabolic action of Antheraea pernyi Guerin-Meneville Pas].
It has been found that the ethyl acetate extract isolated from Antheraea pernyi Pas is able to increase the weight of prostate-semina and levator ani muscle-bulbocavarnosus muscle of castrated mice. In addition, the extract also accelerates the growth of younger male mice and enhances the contents of RNA, DNA and protein in the liver tissue of mice. It has been determined that beta-ecdysone is one of the effective constituents for the androgen-like and anabolic action.
Animals↗
[Nuclear DNA assay in gynecologic malignant tumors and its clinical significance].
This paper analyzes the nuclear DNA content in 80 gynecologic tumors by microspectrophotometry and correlates the results of ovarian malignant tumors with the clinico-pathologic features of the tumor. The DNA content and the percentage of nuclei with over octaploid were significantly higher in gynecologic malignant tumors and ovarian germ cell tumors than in gynecologic benign tumors and ovarian epidermic tumors. The percentage of nuclei with over octaploid was significantly higher in advanced than in early tumors. It seems that DNA content assay may be helpful in the pathological diagnosis of gynecologic tumors.
Adenocarcinoma↗
[Preliminary infection source of downy mildew of Corydalis yanhusuo W. T. Wang].
The downy mildew of Corydalis yanhusuo caused by Peronospora corydalis usually brings about devastating harm in Anhui Province. In the past, it was generally held that the spores in diseased remains buried in soil were the main source of preliminary infection. But, according to our test, it is the mycelia in tubers that are mainly the preliminary infections source.
Oomycetes↗
[Effects of chronoacupuncture na ja fa on gastric acid secretion, plasma gastrin and prostaglandin E1 in patients with peptic ulcer].
Observations on gastric acid secretion, plasma gastrin and prostaglandin E1 in patients with peptic ulcer disease were made after giving acupuncture with Na Ja Fa. The relationship between the chosen points and their effects was also discussed so as to provide more evidence to evaluate and practice the traditional chronoacupuncture more accurately. The results of this experiment were: (1) The gastric acid output of patients with peptic ulcer disease was decreased, while the plasma gastrin and prostaglandin E1 were increased after puncturing with Na Ja Fa. This reveals that the decrease of acid output was not caused by the change of plasma gastrin, however the plasma prostaglandin E1 may be involved in this process. (2) By using points on Stomach and Spleen meridians, there was a better inhibiting effect in acid output than treating the points of other meridians. This showed that using chronoacupuncture should include choosing points according to differentiation and only by laying stress on the relative specialization of the actions of these points one could expect improvement in efficiency. (3) There were no obvious differences between the standard opening points and the group of points which changed to opening points by Dr Shan Yu Tang. This proves that these two groups of points do have some similar functions and are both effective for clinical use.
Acupuncture Therapy↗