Hui Bin Sun

Size-Dependent Positioning of Human Chromosomes in Interphase Nuclei

By using a fluorescence in situ hybridization technique we revealed that for nine different q-arm telomere markers the positioning of chromosomes in human G(1) interphase nuclei was chromosome size-dependent. The q-arm telomeres of large chromosomes are more peripherally located than telomeres on small chromosomes. This highly organized arrangement of chromatin within the human nucleus was discovered by determining the x and y coordinates of the hybridization sites and calculating the root-mean-square radial distance to the nuclear centers in human fibroblasts. We demonstrate here that global organization within the G(1) interphase nucleus is affected by one of the most fundamental physical quantities-chromosome size or mass-and propose two biophysical models, a volume exclusion model and a mitotic preset model, to explain our finding.

Reduction of cytokine-induced expression and activity of MMP-1 and MMP-13 by mechanical strain in MH7A rheumatoid synovial cells

Excessive mechanical load induces harmful outcomes for joint diseases, such as osteoarthritis and rheumatoid arthritis, but physical stimuli at appropriate intensity are essential for growth and maintenance of bone and articular cartilage. Using a fibroblast-like synoviocyte cell line derived from a patient with rheumatoid arthritis, we examined the effects of gentle cyclic strain, focusing on the expression and activity of matrix metalloproteinase-1 (MMP-1) and MMP-13. Synovial cells were cultured on a collagen-coated agar block and exposed to 2% cyclic strain at 6 rev./min for 1 h. Expression of MMP-1 and MMP-13 was assayed using semi-quantitative and real-time PCR, as well as immunoblotting. Their activity was measured using spectrofluorometry and zymography. The results showed that the cyclic strain reduced the mRNA and protein levels of MMP-1 and MMP-13, and that both collagenase and gelatinase activity was decreased under the strain. The reduction in MMP activity by the cyclic strain was not achieved by the transcriptional inhibitor, actinomycin D. In the presence of proinflammatory cytokines, such as IL-1 beta and TNF-alpha, the strain reduced the cytokine-induced expression and activities of MMPs. Interestingly, the strain elevated the mRNA level of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. These results support a potential role of mechanical strain in down-regulating the cytokine-mediated proteolytic processes in synoviocytes.

Correlated Positioning of Homologous Chromosomes in Daughter Fibroblast Cells

A new method of analyzing chromosome segregation in pairs of daughter human fibroblasts revealed that the positioning of chromosomes in daughter nuclei was closely correlated with their relative positions during the G1 interphase. Two topographic values, namely distance and the angular separation between a pair of homologous chromosomes, were determined using fluorescence in-situ hybridization with four different centromeric DNA probes. These topographical values exhibited a broad distribution as a population, but, to our surprise, both were strongly correlated within each pair of cells derived from the same mother cell (daughters). This correlation was not affected by cell-to-cell distances between daughter cells. We demonstrate in this report that the positioning of chromosomes at G1 interphase is chiefly determined by their configuration at mitosis, consistent with the nuclear architecture model in which chromosomes are immobile at a global scale in the G1 interphase nuclei.

Expression and activities of matrix metalloproteinases under oscillatory shear in IL-1-stimulated synovial cells.

Matrix metalloproteinases (MMPs) are known to play a critical role in tissue disintegration, and an elevated level of MMPs is observed in synovium and synovial fluid of joints with rheumatoid arthritis. During joint movement, synovial tissue receives various mechanical stimuli, but effects of mechanical challenges on regulation of MMPs in rheumatic synovium are poorly understood. Focusing on cellular responses to oscillatory fluid shear in human synovial cells, we determined the expression of MMP-1 and MMP-13 by polymerase chain reaction and immunoblotting as well as proteolytic activities of total MMPs by a fibril degradation assay and zymography. The results revealed that ~0.5 dyn/cm 2 oscillatory shear at 1 Hz not only reduced an mRNA level and a protein level of MMP-1 and MMP-13, but it also decreased collagenase and gelatinase activities of total MMPs. Furthermore, the induction of the MMP expression and activities by interleukin-1 was suppressed by the oscillatory shear. Interestingly, the oscillatory shear upregulated the mRNA expression of TIMP-1 and TIMP-2. Our results support a potential role of oscillatory shear in regulating expression and activities of MMPs in the presence and the absence of proinflammatory cytokine.

Differential expression of neurexin mRNA in CA1 and CA3 hippocampal neurons in response to ischemic insult.

A short period of cerebral ischemia will trigger a cascade of events leading to neuronal death. In an effort to elucidate molecular mechanisms underlying differential vulnerability of CA1 and CA3 hippocampal neurons to neurodegeneration, we performed a transcriptional analysis of rat hippocampal neurons following transient global ischemia. In response to 15-min ischemia, the mRNA level of neurexins II alpha and III alpha was elevated in CA1 neurons and CA3 neurons, respectively. Interestingly, the up-regulated neurexin III alpha mRNA in postischemic CA3 consisted of the insert corresponding to the fourth splicing site, while the transcripts in postischemic CA1 neurons and control CA3 neurons lacked the insert. The observed tissue specific expression and the splicing pattern suggest functional importance of neurexins in postischemic degeneration of hippocampal neurons.

PROMOTER COMPETITION ASSAY FOR ANALYZING GENE REGULATION IN JOINT TISSUE ENGINEERING

We describe a new biochemical technique, promoter competition assay, for examining the role of cis-acting DNA elements in tissue cultures. Recent advances in tissue engineering permit the culture of a variety of cells. Many tissues are engineered, however, without an appropriate understanding of molecular machinery that regulates gene expression and cellular growth. For elucidating the role of cis-acting regulatory elements in cellular differentiation and growth, we developed the promoter competition assay. This assay uses a transient transfer into cells of double-stranded DNA fragments consisting of cis-acting regulatory elements. The transferred DNA fragments act as a competitor and titrate the function of their genomic counterparts. Using synovial cells derived from a rheumatoid arthritis patient, we examined a role of NF-kappa B binding sites in the regulation of the expression of matrix metalloproteinase (MMP) genes. The results support a stimulatory role of NF-kappa B in transcriptional regulation of MMP-1 and MMP-13.

Involvement of the calcium-independent receptor for α-latrotoxin in brain ischemia

Abstract Cerebral ischemia is caused by a reduced blood supply to neurons, and vulnerability to neurodegeneration varies considerably among neuronal types. In hippocampus, neurons in the CA1 region are more susceptible to ischemia-induced neuronal death than neurons in the CA3 region, and in response to transient forebrain ischemia a family of calcium-dependent receptors for α-latrotoxin is differentially expressed in the two regions. Here, we report that an ischemic insult up-regulated a family of calcium-independent receptors for α-latrotoxin (CIRL) mRNAs in CA1 neurons and down-regulated their mRNAs in CA3 neurons. Furthermore, antisense oligonucleotides complementary to CIRL-1 mRNA or CIRL-3 mRNA suppressed neuronal death associated with hypoxia in hippocampal and cortical cell cultures. The observed region-specific CIRL mRNA expression in hippocampus and an in vitro rescue experiment by antisense oligonucleotides against CIRL mRNAs suggest a functional importance of CIRL in neurodegeneration.

Model-based analysis of matrix metalloproteinase expression under mechanical shear.

AbstractWe report the development of a model-based analysis for identification of the role of transcription factor (TF) binding motifs. A nonlinear mathematical model was formulated to establish the quantitative relationship between the temporal expression profiles and the distribution of known TF binding motifs on regulatory DNA regions. In order to evaluate biological meaning of the nonlinear model, the role of TF binding motifs predicted by the model was examined by a promoter competition assay where specific TF binding motifs were inactivated by a transient transfer of the DNA fragments consisting of the TF binding motifs. Using the shear stress responses of a family of matrix metalloproteinases in human synovial cells as a model system, we showed that the nonlinear formulation was able to approximate the experimentally observed expression profile better than the linear formulation, and the stimulatory and inhibitory roles of TF binding motifs extracted from the model were validated by the competition assay. The results support that an integrated usage of the nonlinear model and the biochemical evaluation assay would contribute to identifying critical regulatory DNA elements in mechanical responses in connective tissue.

Regulating gene expression using optimal control theory

We described development of a novel genome-based model-driven strategy useful for regulating eukaryotic gene expression. In order to extract biologically meaningful information from a large volume of mRNA expression data, we built previously a PROmoter-Based Estimation (PROBE) model. The PROBE model allowed us to establish a quantitative relationship between transcription-factor binding motifs in regulatory DNA sequences and mRNA expression levels. Here, we extended PROBE formulation to derive an optimal control law for gene regulation. The responses to shear stress in human synovial cells were chosen as a model biological system, and the system dynamics was identified from the expression pattern of the genes involved in degradation and maintenance of extracellular matrix. In order to suppress the responses to mechanical stimuli, a Ricatti equation was solved and an admissible control law was derived. The approach presented here can be implemented in any biological process, and it would be useful to develop a transcription-mediated strategy for gene therapies and tissue engineering.

Future opportunities for life science programs in space

Most space‐related life science programs are expensive and time‐consuming, requiring international cooperation and resources with trans‐disciplinary expertise. A comprehensive future program in “life sciences in space” needs, therefore, well‐defined research goals and strategies as well as a sound ground‐based program. The first half of this review will describe four key aspects such as the environment in space, previous accomplishments in space (primarily focusing on amphibian embryogenesis), available resources, and recent advances in bioinformatics and biotechnology, whose clear understanding is imperative for defining future directions. The second half of this review will focus on a broad range of interdisciplinary research opportunities currently supported by the National Aeronautics and Space Administration (NASA), National Institute of Health (NIH), and National Science Foundation (NSF). By listing numerous research topics such as alterations in a diffusion‐limited metabolic process, bone loss and skeletal muscle weakness of astronauts, behavioral and cognitive ability in space, life in extreme environment, etc., we will attempt to suggest future opportunities.