Biomedical subjects

Y Z Zheng

Publications and source records attributed to Y Z Zheng.

At least 19 recordsLinked to original sources

PubMed2003

CpG oligodeoxynucleotide enhances immunity against blood-stage malaria infection in mice parenterally immunized with a yeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides.

The 19kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)), an analog of the leading falciparum malaria vaccine candidate, induces protective immunity to challenge infection when formulated with complete/incomplete Freund's adjuvant (CFA/IFA), an adjuvant unsuitable for use in humans. In this study, we investigate Montanide ISA51 and Montanide ISA720 as well as CpG oligodeoxynucleotide (ODN) as adjuvants for induction of immunity to MSP1(19). Mice immunized with MSP1(19) adjuvanted with Montanide ISA51 were protected even though some mice experienced low-grade parasitemia before resolving the infection. Mice immunized with MSP1(19) adjuvanted with Montanide ISA720 showed delayed patent parasitemia with all mice ultimately succumbing to infection. Interestingly, when the synthetic CpG ODN 1826 was included in either Montanide formulation, mice were completely protected with no parasites detected in the blood. MSP1(19)-specific antibodies in MSP1(19)-immunized mice adjuvanted with Montanide ISA51 or Montanide ISA720 showed predominantly IgG1 antibody and low levels of IgG2a. CpG ODN 1826 significantly enhanced both IgG1 and IgG2a antibody responses in Montanide ISA51-adjuvanted mice but significantly enhanced only the IgG2a antibody response in Montanide ISA720-adjuvanted mice. To investigate the relative roles of antibody and CD4(+) T cells in protection, MSP1(19)-immunized mice adjuvanted with Montanide ISA720 and CpG ODN 1826 were depleted of CD4(+) T cells just prior to challenge. Results showed that three of nine immunized/T cell depleted mice died following infection. These results suggest that antibody and CD4(+) T cells are critical for protection following immunization with MSP1(19) adjuvanted with Montanide and CpG ODN and that the formulation of a human malaria vaccine candidate in Montanide ISA720 or ISA51 together with human compatible CpG ODN would be useful for improving efficacy.

Adjuvants, Immunologic↗

PubMed2000

Influence of membrane physical state on the lysosomal proton permeability.

Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane potential with 3,3'-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1, 6-diphenyl-1,3,5-hexatriene. Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol and decreased with rigidifying the membranes by cholesteryl hemisuccinate. The proton permeability increased to the maximum of 42% by the benzyl alcohol treatment and decreased to the minimum of 38.1% by the cholesteryl hemisuccinate treatment. Treating the lysosomes with protonophore CCCP increased the proton permeability by 58%. The effects of the membrane fluidization and rigidification can be reversed by rigidifying the fluidized membranes and fluidizing the rigidified membranes, respectively. The results indicate that the proton permeability of lysosomes increased and decreased with increasing and decreasing their membrane fluidity, respectively. Moreover, the lysosomal proton permeability did not alter further if the changes, either an increase or a decrease, in the fluidity exceeded some amount. The results suggest that the proton permeability of lysosomes can be modulated finitely by the alterations in their membrane physical state.

Animals↗

PubMed2000

Influence of membrane fluidity modifiers on lysosomal osmotic sensitivity.

Since lysosomes are prone to osmotic lysis, we have examined the correlation between their physical state and sensitivity to osmotic challenge, using agents which modify membrane fluidity. The latency loss of beta-hexosaminidase after an incubation in hypotonic sucrose medium was followed under different conditions of membrane fluidity, recorded by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3, 5-hexatriene. Increasing fluidity of the lysosomal membranes with benzyl alcohol (BA) and greater rigidity caused by cholesteryl hemisuccinate (CHS) increased and decreased the enzyme latency loss, respectively. The effects of BA and CHS treatments on osmotic sensitivity were reversible subsequently by reciprocal treatments of the lysosomes with CHS and BA, respectively. The results indicate that the physical state of the membrane does indeed affect lysosomal osmotic stability.

Anesthetics, Local↗

PubMed2000

Prevalence of methylenetetrahydrofolate reductase C677T and its association with arterial and venous thrombosis in the Chinese population.

Moderate hyperhomocysteinaemia (MHH) is associated with arterial and venous thrombosis. A main genetic defect related to MHH is a C to T substitution at nucleotide 677 of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene. A prothrombin 20210A mutation was recently identified as a risk factor for arterial and venous thrombosis. However, studies on the prevalence of mutant MTHFR C677T and prothrombin G20210A and their association with thrombosis were controversial and seldom reported in the Chinese population. We investigated the prevalence of MTHFR C677T and prothrombin G20210A genotypes by polymerase chain reaction (PCR) followed by restriction enzyme digestion in 420 Chinese subjects: 53 with deep venous thrombosis (DVT); 145 with cerebrovascular disease [115 cerebral infarction, 30 cerebral haemorrhage (CH)]; 100 with coronary artery disease (CAD); and 122 control subjects. The prevalence of the mutated MTHFR 677TT genotype and the 677T allele in normal controls was 12.3% and 30.7% respectively, similar to that in Caucasians and Japanese. The mutant 677T homozygotes and alleles were more frequent in patients with DVT than in controls (18.9% vs. 12.3%, 0.01 < P < 0. 025; 48.1% vs. 30.7%, P < 0.005). The relative risk of DVT among the carriers of 677TT and 677T were significantly increased [odds ratios: 3.4, 95% confidence interval (CI) 1.3-9.5, and 3.6, 95% CI 1. 7-7.7, respectively). The mutant MTHFR heterozygous 677C/T carriers were increased in patients with cerebral infarction compared with controls (53.9% vs. 36.9%, 0.01 < P < 0.025). Relative risk of cerebral infarction was 0.96 (95% CI 0.4-2.3) for 677TT homozygotes and 1.99 (95% CI 1.2-3.4) for 677C/T heterozygotes. However, the distribution of the MTHFR TT genotype was less frequent in patients with CAD with coronary artery stenosis of > 50% than in controls (2. 8% vs. 12.3%, 0.025 < P < 0.05). Relative risk of CAD was not increased among the carriers of 677TT and 677T (odds ratios: 0.2, 95% CI 0-1.1, and 0.97, 95% CI 0.5-1.8, respectively). There were no differences in the distribution of the MTHFR genotypes among CH, CAD with coronary artery stenosis of < 50% and controls. The prothrombin 20210A mutation was not found in any patients or controls. These results demonstrated that MTHFR 677T was associated with DVT and cerebral infarction but was less associated with CAD in the Chinese population.

Adolescent↗

PubMed2000

Effects of photoinduced membrane rigidification on the lysosomal permeability to potassium ions.

Mechanism for the photoinduced increase in the lysosomal K+ permeability is still unknown. In this study, we investigated the effect of photodamage-induced membrane rigidification on the lysosomal K+ permeability by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and by monitoring proton leakage with p-nitrophenol. Membrane fluidity was measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Methylene blue-mediated photodamage to lysosomes decreased their membrane fluidity and increased their K+ permeability. The photoinduced increase in the K+ permeability can be reversed by fluidizing the rigidified lysosomal membranes with benzyl alcohol. The results suggest that the membrane rigidification induced by photodamage may increase lysosomal K+ permeability. This conclusion is supported by the observation that rigidifying lysosomal membranes by the treatment with membrane rigidifier cholesteryl hemisuccinate also enhanced the lysosomal K+ permeability.

Animals↗

PubMed1999

Optimized production of recombinant bluetongue core-like particles produced by the baculovirus expression system.

The baculovirus-expression vector system (BEVS) has been widely used for the experimental production of many human and animal single- and multi-unit vaccines, heterologous proteins, and viral insecticides. In this work, the production of recombinant bluetongue virus core-like particles (CLPs), using Sf9 cells in shaker-suspension culture with the SF900 II medium (GIBCO, NY), has been studied. This system involved the simultaneous production of two proteins, VP7 and VP3, and was shown to achieve high volumetric productivities. The key parameters of the time of infection (TOI), and the multiplicity of infection (MOI) were studied. The results show that the peak-volumetric yields and cell-specific yields achieved using low MOIs at low-cell densities were the same as those obtained following infections with a high MOI at high-cell densities. This work establishes the feasibility of using low MOIs in the baculovirus system to produce complex multiprotein particles.

Animals↗

PubMed1999

Molecular characterization of a calponin-like protein from Schistosoma japonicum.

The gene for a Schistosoma japonicum (Philippine strain origin) (Sjp) calponin-like protein has been cloned and characterised. The clone, designated P14, was isolated from a Sjp adult worm lambda ZAP cDNA library by immunoscreening, and was shown to contain a full-length cDNA encoding a 38.3 kDa protein that shared significant sequence similarity to a number of previously reported calponins and 22 kDa smooth-muscle proteins. Northern analysis indicated the P14 transcript was approximately 2.2 kb in both Sjp and Chinese strain S. japonicum (Sjc) adult worms. Southern blot analysis of genomic DNA suggested that several copies of the P14 gene are present in the Sjc and Sjp genomes but only one copy was evident in the S. mansoni (Sm) genome. Western blot analysis indicated that the product of P14 occurs as a 38 kDa protein in adult Sjp worms and homologues are present in adult worms of Sjc and Sm. At least six isoforms, all with a similar molecular size of approximately 38 kDa and isoelectric points ranging from 8.1 to 9.5, were present in adult Sjc worms. The protein was immunolocalized to the muscle of male and female Sjc adult worms. Recombinant protein was expressed in E. coli and purified under denaturing conditions, and in yeast to produce a soluble protein in purified form. The availability of purified, correctly folded protein will allow investigations into its biological functions and potential involvement in host immunity.

Amino Acid Sequence↗

PubMed1999

Quantification of recombinant core-like particles of bluetongue virus using immunosorbent electron microscopy.

Immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (BTV) core-like particles (CLP) in either purified preparations or lysates of recombinant baculovirus-infected cells. The capture antibody was an anti-BTV VP7 monoclonal antibody. The CLP concentration in purified preparations was determined to be 6.6 x 10(15) particles/l. CLP concentration in lysates of recombinant baculovirus-infected cells was determined at various times post-infection and shown to reach a value of 3 x 10(15) particles/l of culture medium at 96 h post-infection. The results indicated that immunosorbent electron microscopy, aided by an improved particle counting method, is a simple, rapid and accurate technique for the quantification of virus and virus-like particles produced in large scale in vitro systems.

Animals↗

PubMed1999

Time course study of the chromosome-type breakage-fusion-bridge cycle in maize.

The B chromosome of maize has been used in a study of dicentric chromosomes. TB-9Sb is a translocation between the B and chromosome 9. The B-9 of TB-9Sb carries 60% of the short arm of 9. For construction of dicentrics, a modified B-9 chromosome was used, B-9-Dp9. It consists of the B-9 chromosome plus a duplicated 9S region attached to the distal end. In meiosis, fold-back pairing and crossing over in the duplicated region gives a chromatid-type dicentric B-9 that subsequently initiates a chromatid-type breakage-fusion-bridge cycle. In the male, it forms a single bridge in anaphase II of meiosis and at the first pollen mitosis. However, the cycle is interrupted by nondisjunction of the B centromere at the second pollen mitosis, which sends the B-9 dicentric to one pole and converts it from a chromatid dicentric to a chromosome dicentric. As expected, the new dicentric undergoes the chromosome-type breakage-fusion-bridge cycle and produces double bridges. A large number of plants with chromosome dicentrics were produced in this way. The presence of double bridges in the root cells of plants with a chromosome dicentric was studied during the first 10 wk of development. It was found that the number of plants and cells showing double bridges declined steadily over the 10-wk period. Several lines of evidence indicate that there was no specific developmental time for dicentric loss. "Healing" of broken chromosomes produced by dicentric breakage accounted for much of the dicentric loss. Healing produced a wide range of derived B-9 chromosomes, some large and some small. A group of minichromosomes found in these experiments probably represents the small end of the scale for B-9 derivatives.

Chromosome Mapping↗

PubMed1997

Free luciferase may acquire a more favorable conformation than ribosome-associated luciferase for its activity expression.

A variant of firefly luciferase in which the C-terminal end was extended with 44 amino acid residues served as a model protein in this study. After transcription and translation in vitro, the enzyme activity was measured when still attached to the ribosome and when released from the ribosome by incubation with RNase A or puromycin. It was found that the C-terminally extended luciferase already had activity when linked to the ribosome, but its activity was greatly increased when released from the ribosome. These results indicate that the luciferase is folded during synthesis on the ribosome; however, some conformational adjustments occur after its release from the ribosome which are required for the full expression of its enzymatic activity.

Amino Acid Substitution↗

PubMed1996

Improved method for counting virus and virus like particles.

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a novel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

Antibodies, Monoclonal↗

PubMed1996

Genetic characterization of protein C deficiency in Japanese subjects using a rapid and nonradioactive method for single-stand conformational polymorphism analysis and a model building.

We studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5'-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phe139Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Arg169Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

Amino Acid Sequence↗

PubMed1995

A point mutation (Arg271-->Cys) of a homozygote for dysfunctional prothrombin, prothrombin Obihiro, which has a region of high sequence variability.

The molecular defect of a congenitally dysfunctional form of prothrombin, prothrombin Obihiro, was identified in a patient with a severe bleeding tendency. He showed reduced fibrinogen clotting activity, despite a normal prothrombin antigen level. Nucleotide sequencing of amplified DNA revealed a C-->T change at nucleotide 7311 of exon VIII of the prothrombin gene. This resulted in the substitution of Arg271 by Cys at the factor Xa cleavage site which precludes normal activation of prothrombin Obihiro by factor Xa and the generation of thrombin. The proband was homozygous for this mutation. In addition, seven new nucleotide changes were identified in the prothrombin gene of this patient by comparison with the published gene sequence. Three polymorphisms at nucleotides 4291, 4298 and 7223 were exclusively identified in the prothrombin gene from Japanese. Particularly in the region containing exon VI and introns E and F (nucleotides 4048-4303), the prothrombin gene proved to be of high-sequence variability.

Base Sequence↗

PubMed1995

[The case-control study of risk factors in primary angle-closure glaucoma].

A case-control study was conducted to determine the risk factors of primary angle-closure glaucoma (PACG). In this study, 103 cases of PACG patients and 95 cases of non-glaucoma controls were investigated. It was found that the risk factors of PACG were family history of glaucoma, irritable temper, short distance between eyes and workplace at work and high blood pressure. The results did not support that cigarette smoking, alcoholic consumption, diabetes mellitus, myopia and blood types were related with PACG.

Adult↗

PubMed1995

Protein C Osaka 10 with aberrant propeptide processing: loss of anticoagulant activity due to an amino acid substitution in the protein C precursor.

We studied the molecular basis of protein C deficiency in a family with a history of thromboembolic disease. An approximately 50% reduction in anticoagulant activity despite normal levels of protein C amidolytic activity and antigen was detected in plasma from the proband. All the exons and intron/exon junctions of the protein C gene were studied using a strategy that combined polymerase chain reaction amplification with DNA sequencing of the amplified fragments. We identified a C-to-A change at nucleotide number 1387 of the protein C gene in the proband and his mother, and this mutant was designated protein C Osaka 10. The C-to-A change resulted in the substitution of Ser for Arg at position -1, which is the processing protease cleavage site. The mutant protein C was partially purified from plasma of the patient's mother using barium adsorption followed by ion-exchange column chromatography. It eluted at the same sodium chloride concentration as normal protein C, and thus gamma-carboxylation of the mutant protein appeared to be normal. The apparent molecular weight of this mutant protein C was the same as that of the normal protein on immunoblotting. Amino-terminal sequence analysis showed that the light chain of the mutant protein C had an additional Ser at position-1. Thus, the loss of anticoagulant activity of protein C Osaka 10 can be explained by alteration of the conformation of the Gla domain by the additional Ser in the mutant molecule.

Adult↗

PubMed1994

Six missense mutations associated with type I and type II protein C deficiency and implications obtained from molecular modelling.

The molecular basis of protein C deficiency was studied in three type I and three type II heterozygotes. Three probands showed thrombotic complications. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining by the polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, and DNA sequencing of the PCR-amplified fragments. Six missense mutations were identified, including three novel ones. One was located in exon II, in which the initiating translation codon (ATG) encoding for Met at position -42 was replaced by ACG encoding for Thr. The other five were located in exon IX, and included TAC(Tyr399)-->CAC(His), CCG(Pro327)-->CTG(Leu), GAC(Asp359)-->AAC(Asn) in two cases, and GGG(Gly350)-->AGG(Arg). Four of the six missense mutations occurred in CG dinucleotide. Sequence analysis of the other exons excluded additional mutations. By restriction enzyme analysis, co-segregation of the mutation with protein C deficiency was observed in four families. The other two mutations at amino acid positions -42 and 350 were also considered to be associated with protein C deficiency due to the absence of these mutations in 50 normal individuals. A structural model of the protease domain of mutant activated protein C was constructed by the chimeric modelling method, and the resultant model suggested conformational changes due to each missense mutation identified in protein C deficiency. The present data also provide some evidence regarding the genetic heterogeneity of protein C deficiency.

Adult↗

PubMed1994

[External trabeculotomy in the treatment of developmental glaucoma: a clinical report on 140 eyes of 88 cases].

140 eyes of 88 cases with developmental glaucoma including 128 eyes of 80 cases with infantile glaucoma and 12 eyes of 8 cases with juvenile glaucoma were treated by external trabeculotomy. The success rate of the operation is 73.9% and by addition of small amount of topical antiglaucoma agent the rate of glaucomatous control reaches to 86.4%, the average period of follow-up being 15.2 months. The complications of the operation are few and mild in degree. The important steps of the operative procedure and the critical points of the post-operative management were described, the therapeutic effect of the operation was evaluated and the factors which possibly can affect the prognosis of the operation were analyzed. It is recognized that the operation is better than goniotomy. We recommend external trabeculotomy for treatment of developmental glaucoma which is especially suitable for primary infantile glaucoma.

Adolescent↗

PubMed1994

Three missense mutations in the protein C heavy chain causing type I and type II protein C deficiency.

We have studied the molecular basis of protein C deficiency in three families with a history of thromboembolic disease. An approximately 50% reduction in both functional and immunologic levels of protein C was detected in the plasma from two unrelated patients, designated protein C Osaka 1 and protein C Osaka 2. An approximately 50% reduction in functional level but normal immunologic level of protein C was detected in plasma from a third patient, designated protein C Osaka 3. DNA sequencing of the amplified DNA revealed one missense mutation in each case. Additional mutations in the coding sequence were excluded by DNA sequencing of all protein C exons. We identified a C-to-T change at nucleotide number 6,218 of the protein C gene in protein C Osaka 1. This results in the amino acid substitution of Arg-169 by Trp at the alpha-thrombin cleavage site. In protein C Osaka 2, a G-to-A change at nucleotide number 8,807 was identified leading to the amino acid substitution of Met-364 by Ile in the protease domain. This substitution may impair the synthesis or stability of protein C Osaka 2. In protein C Osaka 3, a G-to-C change at nucleotide number 8,868 was identified. This results in substitution of Gly-385 by Arg in the protease domain. Based on these, it was concluded that Arg-169-to-Trp mutation and Met-364-to-Ile mutation cause type I protein C deficiency and Gly-385-to-Arg mutation causes type II deficiency.

Adult↗