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Biomedical subjects

Heng Zhu

Publications and source records attributed to Heng Zhu.

15 recordsLinked to original sources
PubMed2006

Protein chip fabrication by capture of nascent polypeptides.

The most challenging step in protein microarray fabrication is high-throughput production of proteins. Here we report two similar strategies to fabricate protein chips through capture onto a solid surface of the nascent polypeptides during translation of synthetic or in vitro-transcribed RNAs. Using these approaches, we efficiently fabricated both peptide and protein microarrays at relatively high density. We further demonstrated that such protein chips can be used to analyze protein activity.

Cell-Free System↗
PubMed2006

Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray.

To monitor severe acute respiratory syndrome (SARS) infection, a coronavirus protein microarray that harbors proteins from SARS coronavirus (SARS-CoV) and five additional coronaviruses was constructed. These microarrays were used to screen approximately 400 Canadian sera from the SARS outbreak, including samples from confirmed SARS-CoV cases, respiratory illness patients, and healthcare professionals. A computer algorithm that uses multiple classifiers to predict samples from SARS patients was developed and used to predict 206 sera from Chinese fever patients. The test assigned patients into two distinct groups: those with antibodies to SARS-CoV and those without. The microarray also identified patients with sera reactive against other coronavirus proteins. Our results correlated well with an indirect immunofluorescence test and demonstrated that viral infection can be monitored for many months after infection. We show that protein microarrays can serve as a rapid, sensitive, and simple tool for large-scale identification of viral-specific antibodies in sera.

Algorithms↗
PubMed2005

Global analysis of protein phosphorylation in yeast.

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Eukaryotic Cells↗
PubMed2005

Global analysis of protein function using protein microarrays.

Protein microarrays containing thousands of proteins arrayed at high density can be prepared and probed for a wide variety of activities, thereby allowing the large scale analysis of many proteins simultaneously. In addition to identifying the activities of many previously uncharacterized proteins, protein microarrays can reveal new activities of well-characterized proteins, thus providing new insights about the functions of these proteins. Below, we describe the construction and use of protein microarrays and their applications using yeast as a model system.

Animals↗
PubMed2005

A mathematical model for tear drainage through the canaliculi.

PURPOSE: Tear drainage through the canaliculi has been extensively studied experimentally but there has been no attempt to develop a quantitative model for this process. In this paper, we develop a mathematical model for the tear drainage through the canaliculi. METHODS: The mathematical model is based on the experimental findings of Doane, according to which the muscle action during a blink drives the tear drainage. In this paper, mathematical models are developed for the tear flow and the canalicular deformation, and the model equations are solved to predict the tear drainage rates. RESULTS: The drainage rates depend on various physiological parameters. The time to attain a steady state during the drainage process can vary from about 0.0010 s to 0.0546 s, and the tear drainage rate can vary from 0.10 microl/min to 4.00 microl/min for a normal tear film, for physiologically reasonable values of various system parameters. CONCLUSIONS: The model predictions agree with various physiological experiments, at least qualitatively. The model also helps resolve the differences between various tear drainage experiments.

Blinking↗
PubMed2005

A mathematical model for ocular tear and solute balance.

PURPOSE: In this paper we develop a mathematical model that can predict the steady-state tear film thickness and the dynamic tear film thickness and the solute concentration after instillation of a solute-laden fluid in the eye. METHODS: The mathematical model developed in this paper is based on a balance between the inflow and outflow of tears into the tear film. It incorporates a tear drainage model and a model that relates the tear film thickness to the meniscus radius of curvature. To predict the solute concentrations, the tear balance is coupled with the solute balance. The differential equations for the unsteady balances are solved numerically. RESULTS: The model predicts that the tear film thickness depends on a number of physiological factors, such as rates of tear production and evaporation, geometry and modulus of the canaliculi, and surface tension and viscosity of tears, and varies from about 3 to 15 microm. The model also predicts that the drainage time for an instilled volume of 15 microl is 1283 s. Additionally, the time required for the tracer concentration to decay to 1% of the value immediately after instillation of a drug-laden 40 microl drop is about 2480 s. Similarly, the time for intensity decay for a radioactive tracer after 25 microl instillation is about 1566 s. Also, the model predicts that the fraction of the instilled drug that reaches the cornea is about 1.3% for topical application of timolol. CONCLUSIONS: The predicted results agree reasonably with the reported experimental results, at least qualitatively. The model developed here can serve as a useful tool to develop a more precise understanding of various issues related to tears and also evaluate the effect of various parameters on the tear volume.

Eye↗
PubMed2004

Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips.

The TOR (target of rapamycin) proteins play important roles in nutrient signaling in eukaryotic cells. Rapamycin treatment induces a state reminiscent of the nutrient starvation response, often resulting in growth inhibition. Using a chemical genetic modifier screen, we identified two classes of small molecules, small-molecule inhibitors of rapamycin (SMIRs) and small-molecule enhancers of rapamycin (SMERs), that suppress and augment, respectively, rapamycin's effect in the yeast Saccharomyces cerevisiae. Probing proteome chips with biotinylated SMIRs revealed putative intracellular target proteins, including Tep1p, a homolog of the mammalian PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor, and Ybr077cp (Nir1p), a protein of previously unknown function that we show to be a component of the TOR signaling network. Both SMIR target proteins are associated with PI(3,4)P2, suggesting a mechanism of regulation of the TOR pathway involving phosphatidylinositides. Our results illustrate the combined use of chemical genetics and proteomics in biological discovery and map a path for creating useful therapeutics for treating human diseases involving the TOR pathway, such as diabetes and cancer.

Humans↗
PubMed2004

Regulation of gene expression by a metabolic enzyme.

Gene expression in eukaryotes is normally believed to be controlled by transcriptional regulators that activate genes encoding structural proteins and enzymes. To identify previously unrecognized DNA binding activities, a yeast proteome microarray was screened with DNA probes; Arg5,6, a well-characterized mitochondrial enzyme involved in arginine biosynthesis, was identified. Chromatin immunoprecipitation experiments revealed that Arg5,6 is associated with specific nuclear and mitochondrial loci in vivo, and Arg5,6 binds to specific fragments in vitro. Deletion of Arg5,6 causes altered transcript levels of both nuclear and mitochondrial target genes. These results indicate that metabolic enzymes can directly regulate eukaryotic gene expression.

Aldehyde Oxidoreductases↗
PubMed2003

Negative regulation of calcineurin signaling by Hrr25p, a yeast homolog of casein kinase I.

Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase required for Saccharomyces cerevisiae to respond to a variety of environmental stresses. Calcineurin promotes cell survival during stress by dephosphorylating and activating the Zn-finger transcription factor Crz1p/Tcn1p. Using a high-throughput assay, we screened 119 yeast kinases for their ability to phosphorylate Crz1p in vitro and identified the casein kinase I homolog Hrr25p. Here we show that Hrr25p negatively regulates Crz1p activity and nuclear localization in vivo. Hrr25p binds to and phosphorylates Crz1p in vitro and in vivo. Overexpression of Hrr25p decreases Crz1p-dependent transcription and antagonizes its Ca2+-induced nuclear accumulation. In the absence of Hrr25p, activation of Crz1p by Ca2+/calcineurin is potentiated. These findings represent the first identification of a negative regulator for Crz1p, and establish a novel physiological role for Hrr25p in antagonizing calcineurin signaling.

Calcineurin↗
PubMed2003

Protein analysis on a proteomic scale.

The long-term challenge of proteomics is enormous: to define the identities, quantities, structures and functions of complete complements of proteins, and to characterize how these properties vary in different cellular contexts. One critical step in tackling this goal is the generation of sets of clones that express a representative of each protein of a proteome in a useful format, followed by the analysis of these sets on a genome-wide basis. Such studies enable genetic, biochemical and cell biological technologies to be applied on a systematic level, leading to the assignment of biochemical activities, the construction of protein arrays, the identification of interactions, and the localization of proteins within cellular compartments.

Animals↗
PubMed2003

Protein chip technology.

Microarray technology has become a crucial tool for large-scale and high-throughput biology. It allows fast, easy and parallel detection of thousands of addressable elements in a single experiment. In the past few years, protein microarray technology has shown its great potential in basic research, diagnostics and drug discovery. It has been applied to analyse antibody-antigen, protein-protein, protein-nucleic-acid, protein-lipid and protein-small-molecule interactions, as well as enzyme-substrate interactions. Recent progress in the field of protein chips includes surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyse entire proteomes.

Protein Array Analysis↗
PubMed2003

The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity.

In order to develop clinical diagnostic tools for rapid detection of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

Antigens, Viral↗
PubMed2003

Proteomics.

Fueled by ever-growing DNA sequence information, proteomics-the large scale analysis of proteins-has become one of the most important disciplines for characterizing gene function, for building functional linkages between protein molecules, and for providing insight into the mechanisms of biological processes in a high-throughput mode. It is now possible to examine the expression of more than 1000 proteins using mass spectrometry technology coupled with various separation methods. High-throughput yeast two-hybrid approaches and analysis of protein complexes using affinity tag purification have yielded valuable protein-protein interaction maps. Large-scale protein tagging and subcellular localization projects have provided considerable information about protein function. Finally, recent developments in protein microarray technology provide a versatile tool to study protein-protein, protein-nucleic acid, protein-lipid, enzyme-substrate, and protein-drug interactions. Other types of microarrays, though not fully developed, also show great potential in diagnostics, protein profiling, and drug identification and validation. This review discusses high-throughput technologies for proteome analysis and their applications. Also discussed are the approaches used for the integrated analysis of the voluminous sets of data generated by proteome analysis conducted on a global scale.

Drug Design↗
PubMed2002

"Omic" approaches for unraveling signaling networks.

Signaling pathways are crucial for cell differentiation and response to cellular environments. Recently, a large number of approaches for the global analysis of genes and proteins have been described. These have provided important new insights into the components of different pathways and the molecular and cellular responses of these pathways. This review covers genomic and proteomic (collectively referred to as "omic") approaches for the global analysis of cell signaling, including gene expression profiling and analysis, protein-protein interaction methods, protein microarrays, mass spectroscopy and gene-disruption and engineering approaches.

Animals↗