A comparison of two methods for the microscopic determination of age at death.

The precision and accuracy of the Kerley and Ahlqvist-Damsten microscopic methods of age determination are compared. Both methods were applied to the same sample of 40 femoral thin sections of documented age at death. The results indicate that (1) both methods can be used with equal precision, as suggested by comparable observer errors; and (2) the Kerley method produces overall more accurate age estimates. The low previously published standard error of the Ahlqvist-Damsten method (6.71 years) apparently results from the uneven age distribution and small size (20) of their sample.     

Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins

To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.     

Synthesis of stromal glycosaminoglycans in response to injury

Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14,42, and 84 days. The tissue and/or discs were removed and labeled with ³⁵S-sulfate for 18 h; GAGs were extracted with 4 M guanidine–HCl. Extracts were chromatographed on Q-Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS–PAGE. The nonbound fractions from the chromatography were assayed for TGF-β using Western blot analysis and for hyaluronic acid using an ¹²⁵I-radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-β that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-β. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling. © 1995 Wiley-Liss, Inc.     

Alternative Splicing Products of the Gene for a Human Nuclear Actin-related Protein,hArpNβ/Baf53, that Encode a Protein Isoform,hArpNβS,in the Cytoplasm

A human nuclear actin-related protein, hArpNβ/Baf53, is a component of chromatin remodeling and histone acetyltransferase complexes. We identified two alternative splicing products of the gene for hArpNβ/Baf53. They encoded a protein isoform, hArpNβS; and its fusion product with green fluorescent protein was to be found in the cytoplasm, not the nucleus. The isoforms may contribute to functional regulation of these complexes.     

Coupling Neuropeptide Levels to Structural Plasticity in Drosophila Clock Neurons

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The Structural Basis for Specific Recognition of H3K14 Acetylation by Sth1 in the RSC Chromatin Remodeling Complex

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RELMα可通过抑制PTEN信号通路来促进气道重塑并增加炎症反应

本研究旨在考察抵抗素样分子-α(resistin-like molecule-α,RELMα)在哮喘小鼠模型和小鼠肺上皮细胞中的表达及对气道重塑和炎症反应的影响。本研究通过卵清蛋白(ovalbumin,OVA)诱导小鼠哮喘模型,并评估了小鼠肺组织中RELMα、collagen I和fibronectin-1的表达。为了研究RELMα对PTEN信号通路的调控作用,本研究利用shRNA-RELMα、pcDNA3.0-RELMα和pcDNA3.0-PTEN转染小鼠肺上皮细胞系TC-1来上调或下调RELMα及PTEN的表达。通过Western blotting检测了TC-1细胞中RELMα、collagenⅠ、fibronectin-1、PTEN、TNF-α、IL-1β和IL-6的表达。研究发现,与对照小鼠相比,OVA致敏的哮喘小鼠的肺组织中RELMα、collagen I和fibronectin-1的表达显著升高。上调RELMα可显著升高collagenⅠ、fibronectin-1、TNF-α、IL-1β和IL-6的表达并抑制PTEN信号通路的活化。上调PTEN则可抑制collagenⅠ、fibronectin-1、TNF-α、IL-1β和IL-6的表达。本研究表明,RELMα在哮喘发病过程中高表达,上调RELMα可抑制PTEN信号通路来促进气道重塑并增加炎症反应。     

Scavenger Receptor Cysteine-Rich domains of Lysyl Oxidase-Like2 regulate endothelial ECM and angiogenesis through non-catalytic scaffolding mechanisms

Lysyl oxidases are major actors of microenvironment and extracellular matrix (ECM) remodeling. These cross-linking enzymes are thus involved in many aspects of physiopathology, including tumor progression, fibrosis and cardiovascular diseases. We have already shown that Lysyl Oxidase-Like 2 (LOXL2) regulates collagen IV deposition by endothelial cells and angiogenesis. We here provide evidence that LOXL2 also affects deposition of other ECM components, including fibronectin, thus altering structural and mechanical properties of the matrix generated by endothelial cells. LOXL2 interacts intracellularly and directly with collagen IV and fibronectin before incorporation into ECM fibrillar structures upon exocytosis, as demonstrated by TIRF time-lapse microscopy. Furthermore, surface plasmon resonance experiments using recombinant scavenger receptor cysteine-rich (SRCR) domains truncated for the catalytic domain demonstrated their direct binding to collagen IV. We thus used directed mutagenesis to investigate the role of LOXL2 catalytic domain. Neither enzyme activity nor catalytic domain were necessary for collagen IV deposition and angiogenesis, whereas the SRCR domains were effective for these processes. Finally, surface coating with recombinant SRCR domains restored deposition of collagen IV by LOXL2-depleted cells. We thus propose that LOXL2 SRCR domains orchestrate scaffolding of the vascular basement membrane and angiogenesis through interactions with collagen IV and fibronectin, independently of the enzymatic cross-linking activity.