Biomedical subjects

Yuzhi Zhang

Publications and source records attributed to Yuzhi Zhang.

5 recordsLinked to original sources

PubMed2004

Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature.

Atherosclerotic lesion localization to regions of disturbed flow within certain arterial geometries, in humans and experimental animals, suggests an important role for local hemodynamic forces in atherogenesis. To explore how endothelial cells (EC) acquire functional/dysfunctional phenotypes in response to vascular region-specific flow patterns, we have used an in vitro dynamic flow system to accurately reproduce arterial shear stress waveforms on cultured human EC and have examined the effects on EC gene expression by using a high-throughput transcriptional profiling approach. The flow patterns in the carotid artery bifurcations of several normal human subjects were characterized by using 3D flow analysis based on actual vascular geometries and blood flow profiles. Two prototypic arterial waveforms, "athero-prone" and "athero-protective," were defined as representative of the wall shear stresses in two distinct regions of the carotid artery (carotid sinus and distal internal carotid artery) that are typically "susceptible" or "resistant," respectively, to atherosclerotic lesion development. These two waveforms were applied to cultured EC, and cDNA microarrays were used to analyze the differential patterns of EC gene expression. In addition, the differential effects of athero-prone vs. athero-protective waveforms were further characterized on several parameters of EC structure and function, including actin cytoskeletal organization, expression and localization of junctional proteins, activation of the NF-kappaB transcriptional pathway, and expression of proinflammatory cytokines and adhesion molecules. These global gene expression patterns and functional data reveal a distinct phenotypic modulation in response to the wall shear stresses present in atherosclerosis-susceptible vs. atherosclerosis-resistant human arterial geometries.

Arteriosclerosis↗

PubMed2004

Inverse PCR-based RFLP scanning identifies low-level mutation signatures in colon cells and tumors.

Detecting the presence and diversity of low-level mutations in human tumors undergoing genomic instability is desirable due to their potential prognostic value and their putative influence on the ability of tumors to resist drug treatment and/or metastasize. However, direct measurement of these genetic alterations in surgical samples has been elusive, because technical hurdles make mutation discovery impractical at low-mutation frequency levels (<10(-2)). Here, we describe inverse PCR-based amplified restriction fragment length polymorphism (iFLP), a new technology that combines inverse PCR, RFLP, and denaturing high-performance liquid chromatography to allow scanning of the genome at several thousand positions per experiment for low-level point mutations. Using iFLP, widespread, low-level mutations at mutation frequency 10(-2)-10(-4) were discovered in genes located on different chromosomes, e.g., OGG1, MSH2, PTEN, beta-catenin, Bcl-2, P21, ATK3, and Braf, in human colon cancer cells that harbor mismatch repair deficiency whereas mismatch repair-proficient cells were mutation free. Application of iFLP to the screening of sporadic colon cancer surgical specimens demonstrated widespread low-level mutations in seven out of 10 samples, but not in their normal tissue counterparts, and predicted the presence of millions of diverse, low-incidence mutations in tumors. Unique low-level mutational signatures were identified for each colon cancer cell line and tumor specimen. iFLP allows the high-throughput discovery and tracing of mutational signatures in human cells, precancerous lesions, and primary or metastatic tumors and the assessment of the number and heterogeneity of low-level mutations in surgical samples.

Base Pair Mismatch↗

PubMed2002

A PCR-based amplification method retaining the quantitative difference between two complex genomes.

With the increasing emergence of genome-wide analysis technologies (including comparative genomic hybridization (CGH), expression profiling on microarrays, differential display (DD), subtractive hybridization, and representational difference analysis (RDA)), there is frequently a need to amplify entire genomes or cDNAs by PCR to obtain enough material for comparisons among target and control samples. A major problem with PCR is that amplification occurs in a nonlinear manner and reproducibility is influenced by stray impurities. As a result, when two complex DNA populations are amplified separately, the quantitative relationship between two genes after amplification is generally not the same as their relation before amplification. Here we describe balanced PCR, a procedure that faithfully retains the difference among corresponding amplified genes by using a simple principle. Two distinct genomic DNA samples are tagged with oligonucleotides containing both a common and a unique DNA sequence. The genomic DNA samples are pooled and amplified in a single PCR tube using the common DNA tag. By mixing the two genomes, PCR loses the ability to discriminate among the different alleles and the influence of impurities is eliminated. The PCR-amplified pooled samples can be separated using the DNA tag unique to each individual genomic DNA sample. The principle of this method has been validated with synthetic DNA, genomic DNA, and cDNA applied on microarrays. By removing the bias of PCR, this method allows a balanced amplification of allelic fragments from two complex DNAs even after three sequential rounds of PCR. This balanced PCR approach should allow genetic analysis in minute laser-microdissected tissues, paraffin-embedded archived material, or single cells.

DNA, Complementary↗

PubMed2002

An amplification and ligation-based method to scan for unknown mutations in DNA.

A new approach is presented for the sensitive and selective scanning for unknown DNA mutations, based on ligation-mediated PCR and the use of the glycosylases TDG and MutY. These two highly selective enzymes together can detect about 70% of commonly observed polymorphisms and mutations in human tumors. DNA is cross-hybridized to form mismatches at the positions of point mutations, de-phosphorylated to eliminate any pre-existing phosphorylated DNA ends, and then exposed to enzymatic treatment to remove mismatched thymidine (TDG) or adenine (MutY). The resulting apurinic/apyrimidinic sites at the position of the mismatches are heat-converted to 5'-phosphate-containing strand breaks, the DNA is denatured, and an oligonucleotide is ligated at the position of the newly created 5'-phosphate-containing DNA ends. The ligated oligonucleotide then participates in a PCR reaction that amplifies exponentially only the mutation-containing fragments. Using this method, A-->G mutations in a p53 (TP53)-containing system, T-->G, G-->A, and C-->A, mutations in the Ku gene (XRCC5), and ATM, gene for a number of patient-derived genomic DNA samples have been successfully screened. This PCR-based assay is capable of detecting one mutated allele in 100 normal alleles and requires 5 to 100 ng of genomic DNA as starting material. The assay allows final visualization of the mutated fragments on a common ethidium gel or biotinylation and use in a capture format, potentially allowing the isolation of diverse mutated DNA fragments simultaneously. This versatile new approach should allow high throughput detection of DNA alterations and application in diverse areas of human mutation research.

Adenine↗

PubMed2002

Ligation of a primer at a mutation: a method to detect low level mutations in DNA.

Detection of low frequency mutations following exposure to mutagens or during the early stages of cancer development is instrumental for risk assessment and molecular diagnosis. We present a sensitive new method to detect trace levels of DNA mutations induced within a large excess of wild-type sequences. The method is based on mutation-induced generation of new restriction enzyme recognition sites. A DNA sequence is amplified from genomic DNA or cDNA using a high fidelity polymerase. The purified PCR product is digested with a restriction enzyme that recognizes the newly generated restriction site, partially dephosphorylated and ligated with an oligonucleotide at the position of the mutation. The ligated oligonucleotide is then utilized in two rounds of PCR to amplify the mutated DNA but not the wild-type allele that contains no restriction site. An A-->T polymorphism in mRNA (tenascin gene, A(2366)-->T, Asn-->Ile) and a G-->A polymorphism in genomic DNA (Ku gene, G(74582)-->A, Val-->Ile), both of which generate a restriction site for the enzyme SAU3A1, demonstrate the application. Eleven patient samples pre-characterized for the G(74582)-->A polymorphism in the repair gene Ku are used to demonstrate the reliability of this approach. This technique quantitatively detects the Ku G-->A polymorphism at a mutant frequency of 1.6x10(-6) relative to the wild-type allele. Mutations in p53 that are frequently induced by mutagens can readily be detected using the present method. As an example, using a second enzyme BbvI, a mutation frequently encountered in human cancers (G(14154)-->A mutation, p53 codon 245, Arg-->Gln) was detected in patient samples. The process does not require radioactivity, utilizes established procedures and overcomes several factors known to produce false positives in RFLP-based assays. The present amplification via primer ligation at the mutation (APRIL-ATM) has potential applications in the detection of mutagen-generated genetic alterations, early detection of tumor marker mutations in bodily discharges and the diagnosis of minimal residual disease.

Adenocarcinoma↗