Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.     

Effects of medium perfusion on matrix production by bovine chondrocytes in three-dimensional collagen sponges

Various culture systems have been used for examining the anabolic and catabolic functions of isolated chondrocytes as well as for tissue engineering purposes. Perfusion or frequent medium change is beneficial for three-dimensional (3D) cultures of many cell types. In this study, bovine articular chondrocytes (bACs) were grown in 3D collagen sponges with or without medium perfusion (0.33 mL/min) for up to 15 days. The influence of medium perfusion was evaluated using markers of cartilage matrix accumulation, synthesis, and gene expression. Metachromatic matrix, collagen type II, and hyaluronan accumulated around the cells within the collagen sponges. Sulfated glycosaminoglycans (S-GAGs) that accumulated in the sponge exposed to nonperfused control were 130% of that in the perfused sponge at day 7. S-GAG accumulation after 15 days in the nonperfused control was 230% more than at day 7 (p < 0.01). (35)S-sulfate incorporation during the final 18 h of culture in the sponge exposed to nonperfusion was 180% greater than that in the perfused sponge (p < 0.01). Quantitative analyses show that at day 7, aggrecan and collagen type II gene expression were 350% and 240% greater, respectively, in the nonperfused culture than in the perfused one. These results indicate that perfused conditions that are beneficial for other cell types inhibit chondrogenesis by articular chondrocytes in 3D culture.     

Association of DJ-1 and parkin mediated by pathogenic DJ-1 mutations and oxidative stress

The identification of rare monogenic forms of Parkinson's disease (PD) has provided tremendous insight into the molecular pathogenesis of this disorder. Heritable mutations in alpha-synuclein, parkin, DJ-1 and PINK1 cause familial forms of PD. In the more common sporadic form of PD, oxidative stress and derangements in mitochondrial complex-I function are considered to play a prominent role in disease pathogenesis. However, the relationship of DJ-1 with other PD-linked genes and oxidative stress has not been explored. Here, we show that pathogenic mutant forms of DJ-1 specifically but differentially associate with parkin, an E3 ubiquitin ligase. Chemical cross-linking shows that pathogenic DJ-1 mutants exhibit impairments in homo-dimer formation, suggesting that parkin may bind to monomeric DJ-1. Parkin fails to specifically ubiquitinate and enhance the degradation of L166P and M26I mutant DJ-1, but instead promotes their stability in cultured cells. The interaction of parkin with L166P DJ-1 may involve a larger protein complex that contains CHIP and Hsp70, perhaps accounting for the lack of parkin-mediated ubiquitination. Oxidative stress also promotes an interaction between DJ-1 and parkin, but this does not result in the ubiquitination or degradation of DJ-1. Parkin-mediated alterations in DJ-1 protein stability may be pathogenically relevant as DJ-1 levels are dramatically increased in the detergent-insoluble fraction from sporadic PD/DLB brains, but are reduced in the insoluble fraction from parkin-linked autosomal recessive juvenile-onset PD brains. These data potentially link DJ-1 and parkin in a common molecular pathway at multiple levels that may have important implications for understanding the pathogenesis of inherited and sporadic PD.     

Neonatal impact of leukemia inhibitory factor on neurobehavioral development in rats

Cytokines have been implicated in the etiology or pathology of various psychiatric diseases of developmental origin such as autism and schizophrenia. Leukemia inhibitory factor (LIF) is induced by a variety of brain insults and known to have many influences on mature and immature nervous system. Here, we assessed the neurobehavioral and pathological consequences of peripheral administration of LIF in newborn rats. Subcutaneous LIF injection induced STAT3 phosphorylation in many brain regions and increased glial fibrillary acidic protein (GFAP) immunoreactivity in the neocortex, suggesting that LIF had direct effects in the central nervous system. The LIF-treated rats displayed decreased motor activity during juvenile stages, and developed abnormal prepulse inhibition in the acoustic startle test during and after adolescence. They displayed normal learning ability in active avoidance test, however. Brain neuronal structures and startle responses were grossly normal, except for the cortical astrogliosis during neonatal LIF administration. These results indicate that LIF induction in the periphery of the infant has a significant, but discrete impact on neurobehavioral development.     

Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia

Brain ischemia induces a marked response of resident microglia and hematopoietic cells including monocytes/macrophages. The present study was designed to assess the distribution of microglia/macrophages in cerebral ischemia using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). At 24 h after middle cerebral artery occlusion (MCAO), many round-shaped EGFP-positive cells migrated to the ischemic core and peri-infarct area. At 48-72 h after MCAO, irregular round- or oval-shaped EGFP/ionized calcium-binding adapter molecule 1 (Iba 1)-positive cells increased in the transition zone, while many amoeboid-shaped or large-cell-body EGFP/Iba 1-positive cells were increased in number in the innermost area of ischemia. At 7 days after MCAO, many process-bearing ramified shaped EGFP/Iba 1-positive cells were detected in the transition to the peri-infarct area, while phagocytic cells were distributed in the transition to the core area of the infarction. The distribution of these morphologically variable EGFP/Iba 1-positive cells was similar up to 14 days from MCAO. The present study directly showed the migration and distribution of bone marrow-derived monocytes/macrophages and the relationship between resident microglia and infiltrated hematogenous element in ischemic mouse brain. It is important to study the distribution of intrinsic and extrinsic microglia/macrophage in ischemic brain, since such findings may allow the design of appropriate gene-delivery system using exogenous microglia/macrophages to the ischemic brain area.     

Dedifferentiated liposarcoma with chondroblastic osteosarcomatous dedifferentiation

We describe a rare case of dedifferentiated liposarcoma with features resembling chondroblastic osteosarcoma in the dedifferentiated component. The tumor was removed from the left thigh in a 78-year-old male. It consisted of a well-differentiated liposarcoma and an anaplastic component that contained numerous osteoid and cartilaginous tissues surrounded by high-grade spindle cell sarcoma. To our knowledge, only two cases similar to the divergent chondroblastic osteosarcomatous dedifferentiation of this disease have been reported in the literature.     

Feasibility of using decades-old archival tissues in molecular oncology/epidemiology.

Archival tissues are a bountiful resource for various studies. Polymerase chain reaction permits the use of such tissues for molecular biological analyses of disease causation. However, a comprehensive study using a large number of decades-old samples (20 or more years) for molecular oncology/epidemiology has never been shown to be feasible. We have relied upon the unique tumor registry of atomic bomb survivors to show that such studies are possible using 275 hepatocellular carcinoma and 41 skin cancer cases. We used 23 relatively recent thyroid papillary carcinoma cases from persons living in the vicinity of the Chernobyl nuclear reactor accident for comparison. Degradation of DNA is severe in autopsy hepatocellular carcinoma samples but can be compensated for by decreasing the polymerase chain reaction product size. Increasing the amount of DNA that is used by a factor of 8 improved amplification efficiency from approximately 60 to 80%. Age of the samples was not as great a problem as was the source of procurement. The extracted DNA can be used for all types of assays that require polymerase chain reaction amplification, such as restriction fragment length polymorphism, single-strand conformation polymorphism, and direct sequencing.     

Application of Scanning Acoustic Microscopy for Assessing Stress Distribution in Atherosclerotic Plaque

Scanning acoustic microscopy (SAM) was equipped to assess the acoustic properties of normal and atherosclerotic coronary arteries. The SAM image in the atherosclerotic lesion clearly demonstrated that the sound speed was higher than that in the normal intima, and that the variation of elasticity was found within the fibrous cap of the plaque. Young's elastic modulus of each region was calculated and the finite element analysis was applied to derive the stress distribution in these arterial walls. In a case of normal coronary artery, the stress was dominant in the intima and the distribution was rather homogeneous and in a case of atherosclerosis, high stress was concentrated to the relatively soft lesion in the fibrous cap overlying lipid pool. SAM provides information on the physical properties, which cannot be obtained by the optical microscope. The results would help in understanding the pathological features of atherosclerosis. © 2001 Biomedical Engineering Society. PAC01: 8764-t, 8763Df, 8719Xx, 8719Rr