CRISPR/Cas9-mediated disruption of ALLENE OXIDE SYNTHASE results in defective 12-oxo-phytodienoic acid accumulation and reduced defense against spider mite (Tetranychus urticae) in liverwort (Marchantia polymorpha)

Allene oxide synthase (AOS) is a key enzyme involved in the biosynthesis of 12-oxo-phytodienoic acid (OPDA) and jasmonic acid and plays an important role in plant defense against herbivore attacks. In the liverwort, Marchantia polymorpha, we previously identified cytosol-type MpAOS1 and chloroplast-type MpAOS2 that show AOS activities. However, there is no direct evidence to show the subcellular localization of MpAOSs and their contribution to plant defense via OPDA production in M. polymorpha. In this study, we generated M. polymorpha mutants, with the MpAOS1 and MpAOS2 genes disrupted via CRISPR/Cas9-mediated genome editing; the loss of OPDA production was analyzed in double-knockout mutants. On AOS mutants, the survival rate and oviposition of spider mites (Tetranychus urticae) increased relative to those on wild-type plants. Overall, these findings suggest that defense systems via OPDA-signaling pathways in response to spider mites have been established in M. polymorpha.     

Isolation of the amyloid P component from the Engelbreth-Holm-Swarm (EHS) tumor of the mouse

The amyloid P component was isolated from the mouse EHS tumor, a producer of basement membrane-like material. Following collagenase treatment of the tissue homogenate and centrifugation, the supernatant was purified by calcium-dependent binding to agarose, and elution with ethylenediaminetetraacetic acid. Identification of the purified material as the amyloid P component was established by immunodiffusion and electron microscopic appearance as 8.5 nm pentagonal units, frequently assembled into columns. SDS-PAGE gel electrophoresis yielded 25,000 D bands, suggesting that the amyloid P is of the mouse type. It is proposed that the mouse amyloid P component extracted from the tumor is located within the basotubules present in the pericellular matrix.     

Immunochemical studies on dextrans: Microheterogeneity of dextran NRRL B1397 demonstrated by precipitation with concanavalin A

Dextran NRRL B1397 containing C-2 and C-3 branching points was fractionated by successive additions of concanavalin A (Con A). Quantitative experiments of the precipitations of the fractionates with Con A and homologous rabbit anti-dextran, and on inhibition of the precipitin reaction showed that the original dextran sample was composed of a heterogeneous population of dextran molecules. This heterogeneity was confirmed by methylation studies. The fractions were found to have almost the same contents of 1→3 branches but different contents of 1→2 branches. Fractions rich in 1→2 branches reacted well with both a homologous anti-dextran serum and Con A, whereas fractions poor in 1→2 branches either reacted little or not at all with the antiserum or Con A.     

Immunocytochemical identification of amyloid in formalin-fixed paraffin sections

Summary Formalin-fixed paraffin sections of livers, spleens and kidneys from patients with primary, secondary and familial amyloidosis as well as from a casein-induced murine amyloid model were analysed by an immunocy-tochemical (unlabeled antibody enzyme) method utilizing antisera to amyloid-related proteins. All amyloid deposits of all amyloid types showed positive reactions with anti-AP of the respective species. Positive reaction of anti-human AA to human secondary amyloid deposits and of anti-mouse AA to the deposits of casein-induced murine amyloid was also observed, but there was no species cross reactivity. No significant deposition of the reaction products was produced by anti-immunoglobulin light chains on deposits of any amyloid type, or by anti-AA in the tissues from primary or familial amyloidosis. The results indicate that amyloid proteins AA and AP can survive as antigens through routine histologic preparation, that anti-AP can be a universal marker for deposits of any amyloid type within the same species, and that AA-type amyloid can be identified by this method while there may as yet be no feasible universal marker for the AL-type at present.Presented in part at the 64th Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, California, April, 1980     

Evidence for lysophosphatidic acid 1 receptor signaling in the early phase of neuropathic pain mechanisms in experiments using Ki-16425, a lysophosphatidic acid 1 receptor antagonist

Lysophosphatidic acid is a bioactive lipid mediator with neuronal activities. We previously reported a crucial role for lysophosphatidic acid 1 receptor-mediated signaling in neuropathic pain mechanisms. Intrathecal administration of lysophosphatidic acid (1 nmol) induced abnormal pain behaviors, such as thermal hyperalgesia, mechanical allodynia, A-fiber hypersensitization, and C-fiber hyposensitization, all of which were also observed in partial sciatic nerve injury-induced neuropathic pain. Ki-16425 (30 mg/kg, i.p.), a lysophosphatidic acid 1 receptor antagonist, completely blocked lysophosphatidic acid-induced neuropathic pain-like behaviors, when administered 30 min but not 90 min before lysophosphatidic acid injection, suggesting that Ki-16425 is a short-lived inhibitor. The blockade of nerve injury-induced neuropathic pain by Ki-16425 was maximum as late as 3 h after the injury but not after this critical period. The administration of Ki-16425 at 3 h but not at 6 h after injury also blocked neurochemical changes, including up-regulation of voltage-gated calcium channel α₂δ-1 subunit expression in dorsal root ganglion and reduction of substance P expression in the spinal dorsal horn. All of these results using Ki-16425 suggest that lysophosphatidic acid 1 receptor-mediated signaling which underlies the development of neuropathic pain works at an early stage of the critical period after nerve injury.     

The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis

The preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our high-pressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.     

Mammalian Fat and Dachsous cadherins regulate apical membrane organization in the embryonic cerebral cortex

Compartmentalization of the plasma membrane in a cell is fundamental for its proper functions. In this study, we present evidence that mammalian Fat4 and Dachsous1 cadherins regulate the apical plasma membrane organization in the embryonic cerebral cortex. In neural progenitor cells of the cortex, Fat4 and Dachsous1 were concentrated together in a cell–cell contact area positioned more apically than the adherens junction (AJ). These molecules interacted in a heterophilic fashion, affecting their respective protein levels. We further found that Fat4 associated and colocalized with the Pals1 complex. Ultrastructurally, the apical junctions of the progenitor cells comprised the AJ and a stretch of plasma membrane apposition extending apically from the AJ, which positionally corresponded to the Fat4–Dachsous1-positive zone. Depletion of Fat4 or Pals1 abolished this membrane apposition. These results highlight the importance of the Fat4–Dachsous1–Pals1 complex in organizing the apical membrane architecture of neural progenitor cells.     

Desert hedgehog‐patched 2 expression in peripheral nerves during Wallerian degeneration and regeneration

Hedgehog proteins are important in the development of the nervous system. As Desert hedgehog (Dhh) is involved in the development of peripheral nerves and is expressed in adult nerves, it may play a role in the maintenance of adult nerves and degeneration and regeneration after injury. We firstly investigated the Dhh‐receptors, which are expressed in mouse adult nerves. The Dhh receptor patched(ptc)2 was detected in adult sciatic nerves using RT‐PCR, however, ptc1 was undetectable under the same experimental condition. Using RT‐PCR in purified cultures of mouse Schwann cells and fibroblasts, we found ptc2 mRNA in Schwann cells, and at much lower levels, in fibroblasts. By immunohistochemistry, Ptc2 protein was seen on unmyelinated nerve fibers. Then we induced crush injury to the sciatic nerves of wild‐type (WT) and dhh‐null mice and the distal stumps of injured nerves were analyzed morphologically at different time points and expression of dhh and related receptors was also measured by RT‐PCR in WT mice. In dhh‐null mice, degeneration of myelinated fibers was more severe than in WT mice. Furthermore, in regenerated nerves of dhh‐null mice, minifascicular formation was even more extensive than in dhh‐null intact nerves. Both dhh and ptc2 mRNA levels were down‐regulated during the degenerative phase postinjury in WT mice, while levels rose again during the phase of nerve regeneration. These results suggest that the Dhh‐Ptc2 signaling pathway may be involved in the maintenance of adult nerves and may be one of the factors that directly or indirectly determines the response of peripheral nerves to injury. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis

Ras proteins regulate cell growth, death, and differentiation, and it is well established that this functional versatility is accomplished through their different subcellular localizations. Palmitoylated H- and N-Ras are believed to localize at the perinuclear Golgi and plasma membrane (PM). Notably, however, recycling endosomes (REs) also localize to a perinuclear region, which is often indistinguishable from the Golgi. In this study, we show that active palmitoylated Ras proteins mainly localize intracellularly at REs and that REs act as a way station along the post-Golgi exocytic pathway to the PM. H-Ras requires two palmitoyl groups for RE targeting. The lack of either or both palmitoyl groups leads to the mislocalization of the mutant proteins to the endoplasmic reticulum, Golgi apparatus, or the PM. Therefore, we demonstrate that palmitoylation directs Ras proteins to the correct intracellular organelles for trafficking and activity.