Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (K_r) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of K_r range from approximately 2 x 10^4 to approximately 2 x 10^6 for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10^5 to 2 x 10^6 molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., ⩽10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage

The Ultraviolet Imaging Telescope: Instrument and Data Characteristics

The Ultraviolet Imaging Telescope (UIT) was flown as part of the Astro observatory on the Space Shuttle Columbia in December 1990 and again on the Space Shuttle Endeavor in March 1995. Ultraviolet (1200-3300 Angstroms) images of a variety of astronomical objects, with a 40 arcmin field of view and a resolution of about 3 arcsec, were recorded on photographic film. The data recorded during the first flight are available to the astronomical community through the National Space Science Data Center (NSSDC); the data recorded during the second flight will soon be available as well. This paper discusses in detail the design, operation, data reduction, and calibration of UIT, providing the user of the data with information for understanding and using the data. It also provides guidelines for analyzing other astronomical imagery made with image intensifiers and photographic film.Comment: 44 pages, LaTeX, AAS preprint style and EPSF macros, accepted by PAS

Effects of Phosphodiesterase 3A Modulation on Murine Cerebral Microhemorrhages

Background: Cerebral microbleeds (CMB) are MRI-demonstrable cerebral microhemorrhages (CMH) which commonly coexist with ischemic stroke. This creates a challenging therapeutic milieu, and a strategy that simultaneously protects the vessel wall and provides anti-thrombotic activity is an attractive potential approach. Phosphodiesterase 3A (PDE3A) inhibition is known to provide cerebral vessel wall protection combined with anti-thrombotic effects. As an initial step in the development of a therapy that simultaneously treats CMB and ischemic stroke, we hypothesized that inhibition of the PDE3A pathway is protective against CMH development. Methods: The effect of PDE3A pathway inhibition was studied in the inflammation-induced and cerebral amyloid angiopathy (CAA)-associated mouse models of CMH. The PDE3A pathway was modulated using two approaches: genetic deletion of PDE3A and pharmacological inhibition of PDE3A by cilostazol. The effects of PDE3A pathway modulation on H&E- and Prussian blue (PB)-positive CMH development, BBB function (IgG, claudin-5, and fibrinogen), and neuroinflammation (ICAM-1, Iba-1, and GFAP) were investigated. Results: Robust development of CMH in the inflammation-induced and CAA-associated spontaneous mouse models was observed. Inflammation-induced CMH were associated with markers of BBB dysfunction and inflammation, and CAA-associated spontaneous CMH were associated primarily with markers of neuroinflammation. Genetic deletion of the PDE3A gene did not alter BBB function, microglial activation, or CMH development, but significantly reduced endothelial and astrocyte activation in the inflammation-induced CMH mouse model. In the CAA-associated CMH mouse model, PDE3A modulation via pharmacological inhibition by cilostazol did not alter BBB function, neuroinflammation, or CMH development. Conclusions: Modulation of the PDE3A pathway, either by genetic deletion or pharmacological inhibition, does not alter CMH development in an inflammation-induced or in a CAA-associated mouse model of CMH. The role of microglial activation and BBB injury in CMH development warrants further investigation

Aging Exacerbates Development of Cerebral Microbleeds in a Mouse Model

Background: Cerebral microhemorrhages (CMH) are commonly found in the aging brain. CMH are also the neuropathological substrate of cerebral microbleeds (CMB), demonstrated on brain MRI. Recent studies demonstrate the importance of systemic inflammation in CMH development, but the relationships among inflammation, aging, and CMH development are not well-defined. In the current study, we hypothesized that the pathogenesis of inflammation-induced CMH in mice differs by age. Methods: We studied young (3 months, n = 20) and old (18 months, n = 25) C57BL/6 mice injected with low-dose lipopolysaccharide (LPS; 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. Seven days after the first LPS/saline injection, brains were harvested, sectioned, and stained with hematoxylin and eosin (H&E) and Prussian blue (PB) to estimate acute/fresh and sub-acute CMH development, respectively. The relationships between microglial/macrophage activation (ionized calcium-binding adapter molecule-1), astrocyte activation (glial fibrillary acidic protein), blood-brain barrier (BBB) disruption (brain immunoglobulin G), aging, and CMH development were examined using immunohistochemistry. Results: Aging alone did not increase spontaneous H&E-positive CMH development but significantly increased the number, size, and total area of LPS-induced H&E-positive CMH in mice. LPS- and saline-treated aged mice had significantly larger PB-positive CMH compared with young mice, but the total area of PB-positive CMH was increased only in LPS-treated aged mice. Aged mice had significantly increased microglial/macrophage activation, which correlated with H&E- and PB-positive CMH development. Aged mice treated with LPS had significantly increased astrocyte activation and BBB disruption compared with young LPS-treated mice. Conclusions: Aging makes the brain more susceptible to inflammation-induced CMH in mice, and this increase in CMH with aging is associated with microglial/macrophage activation

Influence of Race on the Female Athlete Triad

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A Murine Model of Inflammation-Induced Cerebral Microbleeds

Background: Cerebral microhemorrhages (CMH) are tiny deposits of blood degradation products in the brain and are pathological substrates of cerebral microbleeds. The existing CMH animal models are β-amyloid-, hypoxic brain injury-, or hypertension-induced. Recent evidence shows that CMH develop independently of hypoxic brain injury, hypertension, or amyloid deposition and CMH are associated with normal aging, sepsis, and neurodegenerative conditions. One common factor among the above pathologies is inflammation, and recent clinical studies show a link between systemic inflammation and CMH. Hence, we hypothesize that inflammation induces CMH development and thus, lipopolysaccharide (LPS)-induced CMH may be an appropriate model to study cerebral microbleeds. Methods: Adult C57BL/6 mice were injected with LPS (3 or 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. At 2 or 7 days after the first injection, brains were harvested. Hematoxylin and eosin (H&E) and Prussian blue (PB) were used to stain fresh (acute) hemorrhages and hemosiderin (sub-acute) hemorrhages, respectively. Brain tissue ICAM-1, IgG, Iba1, and GFAP immunohistochemistry were used to examine endothelium activation, blood-brain barrier (BBB) disruption, and neuroinflammation. MRI and fluorescence microscopy were used to further confirm CMH development in this model. Results: LPS-treated mice developed H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. No surface and negligible H&E-positive CMH were observed in saline-treated mice (n = 12). LPS (3 mg/kg; n = 10) produced significantly higher number, size, and area of H&E-positive CMH at 2 days. LPS (1 mg/kg; n = 9) produced robust development of PB-positive CMH at 7 days, with significantly higher number and area compared with saline (n = 9)-treated mice. CMH showed the highest distribution in the cerebellum followed by the sub-cortex and cortex. LPS-induced CMH were predominantly adjacent to cerebral capillaries, and CMH load was associated with indices of brain endothelium activation, BBB disruption, and neuroinflammation. Fluorescence microscopy confirmed the extravasation of red blood cells into the brain parenchyma, and MRI demonstrated the presence of cerebral microbleeds. Conclusions: LPS produced rapid and robust development of H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. The ease of development of both H&E- and PB-positive CMH makes the LPS-induced mouse model suitable to study inflammation-induced CMH

X=Y–ZH compounds as potential 1,3-dipoles. Part 64: Synthesis of highly substituted conformationally restricted and spiro nitropyrrolidines via Ag(I) catalysed azomethine ylide cycloadditions

1,3-Dipolar reactions of imines of both acyclic and cyclic α-amino esters with a range of nitroolefins using a combination of AgOAc or Ag2O with NEt3 are described. In most cases the reactions were highly regio- and stereospecific and endo-cycloadducts were obtained in good yield. However, in a few cases the initially formed cycloadducts underwent base catalysed epimerisation. The stereochemistry of the cycloadducts was assigned from NOE data and established unequivocally in several cases by X-ray crystallography