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Biomedical subjects

D Y Zhang

Publications and source records attributed to D Y Zhang.

At least 19 recordsLinked to original sources
PubMed2004

Pelargonium necrotic spot virus: a new member of the genus Tombusvirus.

A virus isolate from Pelargonium spp., provisionally designated UPEV (unknown pelargonium virus), had isometric particles 31-33 nm in diameter, with a granular surface structure similar to that of viruses in three genera of family Tombusviridae. Immunoelectron microscopy proved that UPEV was serologically distinct from all examined morphologically similar members of the family Tombusviridae. The induced cytopathology was characterized by large cytoplasmic virion aggregates and the formation of multivesicular bodies derived from mitochondria. Analysis of the complete ssRNA genome sequence revealed four open reading frames (ORFs) arranged like those of viruses in the genera Tombusvirus and Aureusvirus. Sequence comparisons indicated that three of the four ORFs had a high identity (52-97% identical amino acids) with the respective ORFs of tombusvirus species, especially with Carnation Italian ringspot virus, but not with those of viruses in other genera in Tombusviridae. On the contrary, UPEV coat protein had a low indentity (36-53% identical amino acids) with that of the aureusvirus Pothos latent virus. The data suggested that UPEV originated in a recombination event between a tombus- and an aureusvirus. According to its original host and symptom expression we proposed the new virus be named Pelargonium necrotic spot virus (PeNSV) and classified it as a distinct and new species in the genus Tombusvirus.

Capsid Proteins↗
PubMed2003

Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.

AIM: To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. METHODS AND RESULTS: Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g-1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g-1 beef. The total time required from beginning to end of the procedure was 12 h. CONCLUSIONS: The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. SIGNIFICANCE AND IMPACT OF THE STUDY: Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.

Animals↗
PubMed2001

Detection of rare DNA targets by isothermal ramification amplification.

We described previously a novel DNA amplification technique, termed ramification amplification (RAM) (Zhang et al., Gene 211 (1998) 277). This method was designed to utilize a circular probe (C-probe) that is covalently linked by a DNA ligase when it hybridizes to a target. Then, a DNA polymerase extends the bound forward primer along the C-probe and continuously displaces a downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to in vivo 'rolling circle' replication of bacteriophage. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex, and resulting in an exponential amplification. Previously, we were able to achieve a significant amplification using phi29 DNA polymerase that has a high processivity and strong displacement activity. However, due to the intrinsic limitations of the polymerase, we only achieved a sensitivity of 10,000 target molecules, which is insufficient for most practical uses. Therefore, we tested several DNA polymerases and found that exo(-) Bst DNA polymerase meets the requirement for high sensitivity. By further improving the assay condition and format, we are able to detect fewer than ten targets in 1 h and to apply successfully this method for detection of Epstein-Barr virus in human lymphoma specimens.

Blotting, Southern↗
PubMed2001

Multiple redundant sequence elements within the fission yeast ura4 replication origin enhancer.

BACKGROUND: Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized. RESULTS: Systematic deletion of consecutive segments of approximately 50, approximately 100 or approximately 150 bp within the enhancer and its adjacent core origin (ars3002) revealed that several of the approximately 50-bp stretches within the enhancer contribute to its function in partially redundant fashion. Other stretches within the enhancer are inhibitory. Some of the stretches within the enhancer proved to be redundant with sequences within core ars3002. Consequently the collection of sequences important for core origin function was found to depend on whether the core origin is assayed in the presence or absence of the enhancer. Some of the important sequences in the core origin and enhancer co-localize with short runs of adenines or thymines, which may serve as binding sites for the fission yeast Origin Recognition Complex (ORC). Others co-localize with matches to consensus sequences commonly found in fission yeast replication origins. CONCLUSIONS: The enhancer within the ura4 origin cluster in fission yeast contains multiple sequence motifs. Many of these stimulate origin function in partially redundant fashion. Some of them resemble motifs also found in core origins. The next step is to identify the proteins that bind to these stimulatory sequences.

Journal Article↗
PubMed2001

Ramification amplification: a novel isothermal DNA amplification method.

We have developed a novel isothermal DNA amplification method with an amplification mechanism quite different from conventional PCR. This method uses a specially designed circular probe (C-probe) in which the 3' and 5' ends are brought together in juxtaposition by hybridization to a target. The two ends are then covalently linked by a T4 DNA ligase in a target-dependent manner, producing a closed DNA circle. In the presence of an excess of primers (forward and reverse primers), a DNA polymerase extends the bound forward primer along the C-probe and displaces the downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to the "rolling circle" replication of bacteriophages in vivo. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. This ramification process continues until all ssDNAs become double-stranded, resulting in an exponential amplification that distinguishes itself from the previously described nonexponential rolling circle amplification. In this report, we prove the principle of ramification amplification. By using a unique bacteriophage DNA polymerase, Ø29 DNA Polymerase, that has an intrinsic high processivity, we are able to achieve significant amplification within 1 hour at 35 degrees C. In addition, we applied this technique for in situ detection of Epstein-Barr viral sequences in Raji cells.

Cell Line↗
PubMed2001

Differential detection of cytomegalovirus immediate-early messenger RNA in clinical samples using ligation-dependent PCR.

BACKGROUND: Cytomegalovirus (CMV) causes life-threatening infections in immunocompromised patients, especially those with acquired immunodeficiency or organ transplants. Therefore, early detection of CMV is important to guide the clinical management of actively infected patients. Because detection of replicative transcripts indicates that the virus is in the process of being replicated in the infected cell, we applied a novel, sensitive, ligation-dependent (LD)-PCR method to detect CMV immediate-early (IE) messenger RNA (mRNA), an indicator of viral replication. METHODS AND RESULTS: Viral mRNAs were released from infected cells by incubation in 5 M guanidinium thiocyanate, and IE mRNAs were captured onto magnetic beads through oligo(dT) capture probes. Two hemiprobes, each containing an IE mRNA-complementary region and a region for PCR primer binding, were captured by binding to the IE mRNA. These hemiprobes, bound on an IE mRNA in juxtaposition to one another, were linked together by a DNA ligase to form a full probe that served as the template for PCR amplification. This approach detected IE mRNAs in CMV-propagating cells, but not in supernatants containing only viral DNAs. Thirty-one clinical specimens were tested by LD-PCR; 18 specimens were positive (ten specimens, bronchoalveolar lavage [BAL]; five specimens, urine; two specimens, blood; one specimen, biopsy), 17 of which were confirmed by culture. Three culture-positive samples (two specimens, urine; one specimen, BAL) were missed by LD-PCR, and one urine sample was positive by LD-PCR but negative by culture. CONCLUSION: LD-PCR assay is a reliable test for the early diagnosis of active CMV infection in patient specimens.

Cytomegalovirus↗
PubMed2001

Nonimmune hydrops fetalis and activation of the renin-angiotensin system after asphyxia in preterm fetal sheep.

This study examined the hypothesis that the development of hydrops fetalis after asphyxia in the 0.6 gestation sheep fetus would be associated with activation of the fetal renin-angiotensin system (RAS). Fetuses were randomly assigned to either sham occlusion (n = 7) or to 30 min of asphyxia induced by complete umbilical cord occlusion for 30 min (n = 8). Asphyxia led to severe bradycardia and hypotension that resolved after release of occlusion. After occlusion, plasma renin concentration was significantly increased in the asphyxia group compared with controls (P < 0.005) after 3 min (16.3 +/- 5.3 vs. 4.1 +/- 1.3 ng. ml(-1). h(-1)), and 72 h (30.6 +/- 6.3 vs. 3.7 +/- 1.2 ng. ml(-1). h(-1)). Renal renin concentrations and mRNA levels were significantly greater in the asphyxia group after 72 h of recovery. All fetuses in the asphyxia group showed generalized tissue edema, ascites, and pleural effusions after 72 h of recovery. In conclusion, asphyxia in the preterm fetus caused sustained activation of the RAS, which was associated with hydrops fetalis.

Animals↗
PubMed2001

[Studies of mtDNA haplotype polymorphism of Rongcheng population in China].

The loss of one copy of two 9-bp repeats in mtDNA non-coding region V is often found in Asia and Pacific populations. Two hundred and ten samples from Rongcheng county, Shandong province were detected with the deletion frequency of 12.4%. A distribution map of frequencies of mtDNA 9-bp deletion among Asian and Pacific population was made with reference to other published data. Further discussion was made for the hypotheses of affinity and original model of those populations. PCR-RFLP was conducted to obtain the mtDNA polymorphism information in five other mtDNA regions except the 9-bp deletion in 95 samples. Twenty-seven different mtDNA haplotypes were found, and the relationships among these haplotypes have been analyzed by using MEGA2.0 and PHYLIP 3.57. Two new RFLP sites caused by point mutation were also found, which have not been reported in Chinese populations.

Adolescent↗
PubMed2001

[Somatic embryogenesis and plant regeneration in vitro from young shoots of Aralia elate (Miq.) Seem].

Explants excised from the young shoots of Aralia elata (Miq.) Seem. were cultured on MS media. Calli were induced from the explants on MS medium supplemented with 0.5 mg/L 2, 4-D, 0.5 mg/L BA and 0.5 mg/L NAA. Then these calli were transferred onto the MS medium containing 2.0 mg/L 2,4-D + 0.5 mg/L BA + 0.5 mg/L NAA and 0.2% activated charcoal. Under these conditions the somatic embryoids were observed and regenerated plants were obtained from somatic embryogenesis. Then, a experimental system with stability and high regenerating efficiency has been set up for the propagation of the young plants, the cell breeding technology and the control of somatic embryogenesis of Aralia elata (Miq.).

Aralia↗
PubMed2000

Intramolecular interaction of yeast TFIIB in transcription control.

The general transcription factor TFIIB is a key component in the eukaryotic RNA polymerase II (RNAPII) transcriptional machinery. We have previously shown that a yeast TFIIB mutant (called YR1m4) with four amino acid residues in a species-specific region changed to corresponding human residues affects the expression of genes activated by different activators in vivo. We report here that YR1m4 can interact with several affected activators in vitro. In addition, YR1m4 and other mutants with amino acid alterations within the same region can interact with TATA-binding protein (TBP) and RNAPII normally. However, YR1m4 is defective in supporting activator-independent transcription in assays con-ducted both in vitro and in vivo. We further demonstrate that the interaction between the C-terminal core domain and the N-terminal region is weakened in YR1m4 and other related TFIIB mutants. These results suggest that the intramolecular interaction property of yeast TFIIB plays an important role in transcription regulation in cells.

DNA-Binding Proteins↗
PubMed2000

Serum hepatitis C virus RNA levels and histologic findings in liver allografts with early recurrent hepatitis C.

BACKGROUND: Histopathologic features of early recurrent hepatitis C after orthotopic liver transplantation (OLTx) may be modified by immunosuppressive therapy or complicated by other conditions. Hepatitis C virus (HCV) RNA level usually increases after OLTx, but its correlation to histologic findings is not clear. OBJECTIVE: To evaluate the histologic findings of early recurrent hepatitis C in liver allografts and its correlation to serum HCV RNA level. METHODS: We studied 14 patients who underwent OLTx for chronic HCV infection. Thirty liver biopsy specimens and HCV RNA levels of 22 corresponding plasma samples obtained during the first 6 months following OLTx were analyzed. The control group (9 patients, 25 biopsy specimens) was chosen at random from patients with chronic liver disease other than HCV who were undergoing OLTx, and all tested negative for HCV RNA by polymerase chain reaction after OLTx. RESULTS: Statistically significant pathological features of early recurrent HCV infection were the number of acidophilic bodies, piecemeal necrosis, lymphocyte predominance in the portal tracts, and fibrous septum. These findings and histologic activity index scores increased with time after OLTx. The HCV RNA levels determined by branched DNA assay showed no significant correlation with histologic features. However, patients with higher histologic activity index scores tended to have higher RNA levels. CONCLUSIONS: Liver biopsy specimens are helpful for the diagnosis or confirmation of early recurrent hepatitis C in liver allografts, but serial biopsy specimens are sometimes required for definite diagnosis. The HCV RNA levels are usually higher in patients who display signs of more severe liver damage.

Biopsy↗
PubMed2000

Effects of placental insufficiency on the ovine fetal renin-angiotensin system.

We postulated that chronic placental insufficiency would be associated with reduced expression of renal renin and angiotensinogen genes in the fetal sheep. Placental development was restricted in ewes by removing the majority of caruncles prior to mating (placentally restricted (PR) group). The weights of PR fetuses were significantly reduced (P < 0.05, 2.98 +/- 0.33 kg) compared to control fetuses (4.20 +/- 0.30 kg). Kidney weights were also significantly reduced in the PR fetuses (P < 0.05, 8.4 +/- 0.9 g) compared with control fetuses (12.2 +/- 1.3 g). The ratios of renal renin/-actin mRNA levels were significantly reduced in PR fetuses (P < 0.001, 0.35 +/- 0.02) when compared to control animals (0.98 +/- 0.13). The renal angiotensinogen mRNA/18S rRNA ratio was significantly lower (P < 0.05, 0.28 +/- 0.13) in PR fetuses compared with control fetuses (0.72 +/- 0.10), while hepatic angiotensinogen was unaffected. There was a positive correlation between renal renin mRNA and renal angiotensinogen mRNA levels (r = 0.65, P < 0.05, n = 12). It is unlikely that these changes in renal angiotensinogen and renin mRNA were due to the small increment in plasma cortisol levels (< 5 nmol l-1). There was, however, a positive correlation between arterial PO2 and renal renin mRNA (r2 = 0.77, P < 0.01). Plasma renin levels were not different between the two groups. Thus, restriction of nutrient and oxygen supply throughout fetal life was associated with suppression of renal renin and renal angiotensinogen gene expression, with no effect on hepatic angiotensinogen mRNA levels. This specific suppression of fetal renal renin and angiotensinogen expression could alter the activity of the intrarenal RAS and so affect growth and development of the kidney.

Angiotensinogen↗
PubMed2000

[Studies on single cell cloning of Ginkgo biloba L].

OBJECTIVE: To investigate the relations of the productivity stability of to the homogenization of Ginkgo biloba cells by single cell clones. METHOD: Using plant single cell plate culture technique. RESULTS: Adding 500 ml.L-1L-glutamine in the medium increased greatly the cell plating efficiency (PE), and within the range 0.5-5.0 x 10(4) Cell.ml-1 of the initial cell density, the PE increased with the increment of the cell density. Of the 48 cell clones obtained, the GKB content in G-22 clone reached 0.099%; and most of the clones remain stable in growth and GKB production during subcultures. CONCLUSION: During the cell and tissue culture of G. biloba, the application of plant single cell cloning technique helps to a certain extent solve the problem of productivity instability of ginkgolide.

Cell Line↗
PubMed2000

[The dynamic measurements of human body's unbalance].

A through study of the postural stability of 40 subjects has been conducted with a stabilometer and the computer system. The subjects stand on the testing platform for 10 seconds and meanwhile the sum of frequency spectrum is being analyzed. The most men subjects maintain a little sway well within timits during the trials. The testing conditions include open eyes, close eyes, 10 degrees, 20 degrees, 30 degrees of platform inclination, transverse vision tracking, longitudinal vision tracking and vectorial vision tracking, All testing results show that there are unbalance differences between different age groups under different conditions.

Adult↗
PubMed1999

Human papillomavirus may be common within nasopharyngeal carcinoma of Caucasian Americans: investigation of Epstein-Barr virus and human papillomavirus in eastern and western nasopharyngeal carcinoma using ligation-dependent polymerase chain reaction.

BACKGROUND: Nasopharyngeal carcinoma (NPC), particularly those tumors endemic to the Far East, commonly harbor Epstein-Barr virus (EBV), thought to serve as an important oncogenic promoter. Human papillomavirus (HPV) is associated with a proportion of upper aerodigestive tract carcinomas. We hypothesized that HPV might also contribute to the pathogenesis of NPC, and we queried whether geographic and racial distinctions may be identified between NPC of the Far East versus those diagnosed in Caucasian American patients with regard to the interrelationship of histologic subtype and viral infection. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded tissue (FFPET) from 30 patients (6 Caucasian Americans, 1 Chinese American, 14 and 9 patients from Korea and China, respectively) were studied using the ligation-dependent polymerase chain reaction (LD-PCR). These cases were histologically classified according to the World Health Organization (WHO) schema for NPC. Consensus target probes complementary to the L1 region of over 30 HPV types, as well as target probes complementary to EBER-1 (EBV-related nontranslated latency-associated RNA), were used to amplify target sequences. RESULTS: Seven of 30 NPC (23%) contained HPV sequences. There were 6 Caucasian American patients with NPC; 3 cases (50%) were HPV positive (HPV+). Two of these Caucasian Americans had WHO type I tumors: one was HPV+ and EBV negative (EBV-) and the other was HPV-/EBV+. The remaining Caucasian American NPCs were WHO-II/III tumors which tested as follows: two were coinfected with HPV and EBV, the other two contained EBER but not HPV sequences. The single Oriental American patient had a WHO-III NPC which was HPV-/EBV+. Of the Eastern NPC patients, 4 (1 WHO-I, 3 WHO-II/III) of 23 (17%) NPCs contained HPV sequences as well as EBV. Conclusion. Human papillomavirus appears to be uncommonly (17%) associated with NPC in patients from the Far East and was detected more often (50%) in NPC from American Caucasian patients. Some of these tumors conformed to our perceptions and expectations of NPC (eg, WHO-I tumors being EBV-/HPV+ and WHO-III tumors being EBV+/HPV-), but other tumors did not conform to these expectations (eg, WHO-III NPC occasionally harboring both HPV and EBV). There appears to be a broad profile in the relationship between HPV, EBV, and NPC histologic subtype. Unfortunately, the number of American Caucasian cases studied are too small to allow for strong conclusions.

Asian People↗
PubMed1999

Effects of aortic ligation on the renin angiotensin system in hydronephrotic mice.

A model of aortic ligation in mice with a hydronephrotic kidney (absence of macula densa) was used to determine the effects of aortic ligation on the renal renin-angiotensin system (RAS). Blood pressure increased from 83 +/- 2 to 133 +/- 8 mmHg within 7 days after aortic ligation (p < 0.01). Aortic ligation increased plasma renin (p < 0.01); renin (p < 0.05) and renin mRNA levels (p < 0.001) rose in the ischaemic kidney. In mice with a left hydronephrotic kidney without ischaemia, blood pressure did not change significantly. Plasma renin levels from the left renal vein were lower than from the contralateral vein, but renin (p < 0.01) and renin mRNA levels (p < 0.05) in the hydronephrotic kidney were higher than in the contralateral kidney. In mice with hydronephrosis that had an aortic ligature, blood pressure increased from 81 +/- 2 to 135 +/- 6 mmHg (p < 0.01). Plasma renin increased; renin and renin mRNA levels increased significantly in the ischaemic hydronephrotic kidney (p < 0.01), but not in the contralateral kidney. Thus, the presence of the macula densa is critical for renin release but not for renin gene expression. Aortic ligation results in a significant rise in blood pressure and the activity of the RAS. The mechanisms may involve a baroreceptor and/or an unknown factor.

Animals↗
PubMed1998

Amplification of target-specific, ligation-dependent circular probe.

We describe a novel polymerase chain reaction (PCR)-based gene amplification method utilizing a circularizable oligodeoxyribonucleotide probe (C-probe). The C-probe contains two target complementary regions located at each terminus and an interposed generic PCR primer binding region. The hybridization of C-probe to a target brings two termini in direct apposition as the complementary regions of C-probe wind around the target to form a double helix. Subsequent ligation of the two termini results in a covalently linked C-probe that becomes 'locked on to' the target. The circular nature of the C-probe allows for the generation of a multimeric single-stranded DNA (ssDNA) via extension of the antisense primer by Taq DNA polymerase along the C-probe and displacement of downstream strand, analogous to 'rolling circle' replication of bacteriophage in vivo. This multimeric ssDNA then serves as a template for multiple sense primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. Subsequent thermocycling denatures the dsDNA and initiates the next round of primer extension and ramification. This model results in significantly improved amplification kinetics (super-exponential) as compared to conventional PCR. Our results show that the C-probe was 1000 times more sensitive than the corresponding linear hemiprobes for detecting Epstein-Barr virus early RNA. The C-probe not only increases the power of amplification but also offers a means for decontaminating carryover amplicons. As the ligated C-probes possess no free termini, they are resistant to exonuclease digestion, whereas contaminated linear amplicons are susceptible to digestion. Treatment of the ligation reaction mixture with exonuclease prior to amplification eliminated the amplicon contaminant, which could also have been co-amplified with the same PCR primers; only the ligated C-probes were amplified. The combined advantages of the C-probe and thermocycling have a broad applicability for the detection of both DNA and RNA. Finally, we described a novel isothermal amplification method, ramification extension amplification, utilizing circular nature of C-probe and displacement activity of DNA polymerase.

Binding Sites↗