Two-Color Laser Speckle Shift Strain Measurement System

A two color laser speckle shift strain measurement system based on the technique of Yamaguchi was designed. The dual wavelength light output from an Argon Ion laser was coupled into two separate single-mode optical fibers (patchcords). The output of the patchcords is incident on the test specimen (here a structural fiber). Strain on the fiber, in one direction, is produced using an Instron 4502. Shifting interference patterns or speckle patterns will be detected at real-time rates using 2 CCD cameras with image processing performed by a hardware correlator. Strain detected in fibers with diameters from 21 microns to 143 microns is expected to be resolved to 15 mu epsilon. This system was designed to be compact and robust and does not require surface preparation of the structural fibers

TATA-Binding Protein Recognition and Bending of a Consensus Promoter Are Protein Species Dependent

The structure and behavior of full-length human TBP binding the adenovirus major late promoter (AdMLP) have been characterized using biophysical methods. The human protein induces a 97° bend in DNAAdMLP. The high-resolution functional data provide a quantitative energetic and kinetic description of the partial reaction sequence as native human TBP binds rapidly to a consensus promoter with high affinity. The reaction proceeds with successive formation of three bound species, all having strongly bent DNA, with the concurrence of binding and bending demonstrated by both fluorescence and anisotropy stopped flow. These results establish the protein species dependence of the TBP−DNAAdMLP structure and recognition mechanism. Additionally, the strong correlation between the DNA bend angle and transcription efficiency demonstrated previously for yeast TBP is shown to extend to human TBP. The heterologous NH2-terminal domains are the apparent source of the species-specific differences. Together with previous studies the present work establishes that TBPwt−DNATATA function and structure depend both on the TATA box sequence and on the TBP species

TATA-Binding Protein Recognition and Bending of a Consensus Promoter Are Protein Species Dependent

The structure and behavior of full-length human TBP binding the adenovirus major late promoter (AdMLP) have been characterized using biophysical methods. The human protein induces a 97° bend in DNAAdMLP. The high-resolution functional data provide a quantitative energetic and kinetic description of the partial reaction sequence as native human TBP binds rapidly to a consensus promoter with high affinity. The reaction proceeds with successive formation of three bound species, all having strongly bent DNA, with the concurrence of binding and bending demonstrated by both fluorescence and anisotropy stopped flow. These results establish the protein species dependence of the TBP−DNAAdMLP structure and recognition mechanism. Additionally, the strong correlation between the DNA bend angle and transcription efficiency demonstrated previously for yeast TBP is shown to extend to human TBP. The heterologous NH2-terminal domains are the apparent source of the species-specific differences. Together with previous studies the present work establishes that TBPwt−DNATATA function and structure depend both on the TATA box sequence and on the TBP species

Two bovine genes for cytochrome c oxidase subunit IV: a processed pseudogene and an expressed gene

We have isolated and analyzed 17 clones from a bovine genomic library in phage [lambda] Charon28 probed with a bovine liver cDNA for cytochrome c oxidase subunit IV. Restriction enzyme mapping and Southern analysis indicated that these clones represent only two genomic regions. One region was shown by nucleotide sequencing to contain a subunit IV pseudogene of the processed type. The other class of clones contained the 5' region of a putative expressed gene; the region consists of two exons and two introns, with one exon encoding exclusively the domain representing the presequence present on newly synthesized subunit-IV polypeptides. Genomic Southern analysis indicated that these two clones probably represent the only sequences in the bovine nucleus that share nucleotide sequence identity with the liver subunit IV cDNA when utilizing moderately stringent hybridization conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26919/1/0000485.pd

Adeno-Assocated Virus (AAV) Serotype 9 Provides Global Cardiac Gene Transfer Superior to AAV1, AAV6, AAV7, and AAV8 in the Mouse and Rat

Heart disease is the leading cause of morbidity and mortality. Cardiac gene transfer may serve as a novel therapeutic approach. This investigation was undertaken to compare cardiac tropisms of adeno-associated virus (AAV) serotypes 1, 6, 7, 8, and 9. Neonatal mice were injected with 2.5 × 1011 genome copies (GC) of AAV serotype 1, 6, 7, 8, or 9 expressing LacZ under the control of the constitutive chicken β-actin promoter with cytomegalovirus enhancer promoter via intrapericardial injection and monitored for up to 1 year. Adult rats were injected with 5 × 1011 GC of the AAV vectors via direct cardiac injection and monitored for 1 month. Cardiac distribution of LacZ expression was assessed by X-Gal histochemistry, and β-galactosidase activity was quantified in a chemiluminescence assay. Cardiac functional data and biodistribution data were also collected in the rat. AAV9 provided global cardiac gene transfer stable for up to 1 year that was superior to other serotypes. LacZ expression was relatively cardiac specific, and cardiac function was unaffected by gene transfer. AAV9 provides high-level, stable expression in the mouse and rat heart and may provide a simple alternative to the creation of cardiac-specific transgenic mice. AAV9 should be used in rodent cardiac studies and may be the vector of choice for clinical trials of cardiac gene transfer

Identification of a neurovascular signaling pathway regulating seizures in mice

ObjectiveA growing body of evidence suggests that increased blood–brain barrier (BBB) permeability can contribute to the development of seizures. The protease tissue plasminogen activator (tPA) has been shown to promote BBB permeability and susceptibility to seizures. In this study, we examined the pathway regulated by tPA in seizures.MethodsAn experimental model of kainate‐induced seizures was used in genetically modified mice, including mice deficient in tPA (tPA−/−), its inhibitor neuroserpin (Nsp−/−), or both (Nsp:tPA−/−), and in mice conditionally deficient in the platelet‐derived growth factor receptor alpha (PDGFRα).ResultsCompared to wild‐type (WT) mice, Nsp−/− mice have significantly reduced latency to seizure onset and generalization; whereas tPA−/− mice have the opposite phenotype, as do Nsp:tPA−/− mice. Furthermore, interventions that maintain BBB integrity delay seizure propagation, whereas osmotic disruption of the BBB in seizure‐resistant tPA−/− mice dramatically reduces the time to seizure onset and accelerates seizure progression. The phenotypic differences in seizure progression between WT, tPA−/−, and Nsp−/− mice are also observed in electroencephalogram recordings in vivo, but absent in ex vivo electrophysiological recordings where regulation of the BBB is no longer necessary to maintain the extracellular environment. Finally, we demonstrate that these effects on seizure progression are mediated through signaling by PDGFRα on perivascular astrocytes.InterpretationTogether, these data identify a specific molecular pathway involving tPA‐mediated PDGFRα signaling in perivascular astrocytes that regulates seizure progression through control of the BBB. Inhibition of PDGFRα signaling and maintenance of BBB integrity might therefore offer a novel clinical approach for managing seizures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112290/1/acn3209.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/112290/2/acn3209-sup-0001-TableS1.pd

Breast Cancer DNA Methylation Profiles Are Associated with Tumor Size and Alcohol and Folate Intake

Although tumor size and lymph node involvement are the current cornerstones of breast cancer prognosis, they have not been extensively explored in relation to tumor methylation attributes in conjunction with other tumor and patient dietary and hormonal characteristics. Using primary breast tumors from 162 (AJCC stage I–IV) women from the Kaiser Division of Research Pathways Study and the Illumina GoldenGate methylation bead-array platform, we measured 1,413 autosomal CpG loci associated with 773 cancer-related genes and validated select CpG loci with Sequenom EpiTYPER. Tumor grade, size, estrogen and progesterone receptor status, and triple negative status were significantly (Q-values <0.05) associated with altered methylation of 209, 74, 183, 69, and 130 loci, respectively. Unsupervised clustering, using a recursively partitioned mixture model (RPMM), of all autosomal CpG loci revealed eight distinct methylation classes. Methylation class membership was significantly associated with patient race (P<0.02) and tumor size (P<0.001) in univariate tests. Using multinomial logistic regression to adjust for potential confounders, patient age and tumor size, as well as known disease risk factors of alcohol intake and total dietary folate, were all significantly (P<0.0001) associated with methylation class membership. Breast cancer prognostic characteristics and risk-related exposures appear to be associated with gene-specific tumor methylation, as well as overall methylation patterns

A review of the US Global Change Research Program and NASA's Mission to Planet Earth/Earth Observing System

This report reflects the results of a ten-day workshop convened at the Scripps Institution of Oceanography July 19-28, 1995. The workshop was convened as the first phase of a two part review of the U.S. Global Change Research Program (USGCRP). The workshop was organized to provide a review of the scientific foundations and progress to date in the USGCRP and an assessment of the implications of new scientific insights for future USGCRP and Mission to Planet Earth/Earth Observing System (MTPE/EOS) activities; a review of the role of NASA's MTPE/EOS program in the USGCRP observational strategy; a review of the EOS Data and Information System (EOSDIS) as a component of USGCRP data management activities; and an assessment of whether recent developments in the following areas lead to a need to readjust MTPE/EOS plans. Specific consideration was given to: proposed convergence of U.S. environmental satellite systems and programs, evolving international plans for Earth observation systems, advances in technology, and potential expansion of the role of the private sector. The present report summarizes the findings and recommendations developed by the Committee on Global Change Research on the basis of the presentations, background materials, working group deliberations, and plenary discussions of the workshop. In addition, the appendices include summaries prepared by the six working groups convened in the course of the workshop