Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24

Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP) located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.     

Matricellular Protein Cyr61 Bridges Lysophosphatidic Acid and Integrin Pathways Leading to Cell Migration

Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA₁) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA₁ is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6β1 and ανβ3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.     

Ser18 and 23 phosphorylation is required for p53-dependent apoptosis and tumor suppression

Mouse p53 is phosphorylated at Ser18 and Ser23 after DNA damage. To determine whether these two phosphorylation events have synergistic functions in activating p53 responses, we simultaneously introduced Ser18/23 to Ala mutations into the endogenous p53 locus in mice. While partial defects in apoptosis are observed in p53S18A and p53S23A thymocytes exposed to IR, p53-dependent apoptosis is essentially abolished in p53S18/23A thymocytes, indicating that these two events have critical and synergistic roles in activating p53-dependent apoptosis. In addition, p53S18/23A, but not p53S18A, could completely rescue embryonic lethality of Xrcc4(-/-) mice that is caused by massive p53-dependent neuronal apoptosis. However, certain p53-dependent functions, including G1/S checkpoint and cellular senescence, are partially retained in p53(S18/23A) cells. While p53(S18A) mice are not cancer prone, p53S18/23A mice developed a spectrum of malignancies distinct from p53S23A and p53(-/-) mice. Interestingly, Xrcc4(-/-)p53S18/23A mice fail to develop tumors like the pro-B cell lymphomas uniformly developed in Xrcc4(-/-) p53(-/-) animals, but exhibit developmental defects typical of accelerated ageing. Therefore, Ser18 and Ser23 phosphorylation is important for p53-dependent suppression of tumorigenesis in certain physiological context.     

A 23.8-kD alpha-zein with N-terminal sequence and immunological properties similar to 26.7-kD alpha-zeins

A 23.8-kD alpha-zein polypeptide, K55PC7, has been shown to be a truncated member of the 26.7-kD alpha-zein class based on its amino acid composition, N-terminal sequence, and immunological properties. This unusual polypeptide was isolated by chromatographing whole alpha-zein from inbred K55. The N-terminal sequence of K55PC7 is highly homologous to those of 4 putative 26.7-kD alpha-zeins but shows no homology to those of 10 putative alpha-zeins that belong to the 23.8-kD class. Its higher valine and lower phenylalanine contents also suggest that K55PC7 is a member of the 26.7-kD class. In addition, studies with antibodies raised to peptides corresponding to regions unique to each of the two alpha-zein classes indicate that K55PC7 has immunological similarity to 26.7-kD alpha-zeins. Peptide mapping data suggest that K55PC7 is not the putative product of the truncated 26.7-kD alpha-zein gene zA1 isolated from inbred W64A and described by Spena et al. [26]. It appears that K55PC7 occurs as a major component in inbred K55 and is a truncated version of a 26.7-kD alpha-zein, arisen either by an internal deletion or premature termination due to a nonsense mutation.Abbreviations SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - EIF isoelectric focusing - SP sulfopropyl-Sephadex - PC phosphocellulose - DSS disuccinimidyl suberate - MBC m-maleimido-benzoyl-N-hydroxysuccinimide - 2-ME 2-mercaptoethanol     

The plant growth retardant CCC as inhibitor of gibberellin biosynthesis inFusarium moniliforme

Summary Gibberellin (GA) production inFusarium moniliforme (Gibberella fujikuroi) is suppresed by adding the plant growth retardant CCC [(2-chloroethyl)trimethylammonium chloride] to the culture medium. A concentration of 0.1 mg/l of CCC causes 50% inhibition whereas 10 mg/l and higher concentrations fully suppress GA production. Dry weight of the mycelium is not, or only slightly reduced in the presence of CCC.Thin-layer chromatography of acidic fractions of CCC-free cultures reveals fluorescent spots at 4 differentR _f values. No fluorescent spots can be detected on chromatograms of acidic fractions obtained from CCC cultures, thus demonstrating that production of all GA's is inhibited by CCC.If CCC is added to the medium 2 or 3 days after inoculation, further GA production is blocked, but the level of GA present at the time of CCC application is maintained. CCC does not enhance inactivation of GA₃ in sterile culture medium, nor in the presence of the fungus. It is therefore concluded that CCC inhibits the biosynthesis of GA in the fungus.Transfer of thoroughly washed mycelium from medium with CCC to fresh medium does not result in GA production because sufficient CCC is carried over in the mycelium to block GA biosynthesis completely.     

Inhibition of cell division by blue light

Microsporocytes of Lilium and Trillium can revert into a mitotic cycle when separated from the anther before the beginning of meiosis and cultured in vitro. Repeated short daily irradiations with visible blue light (10⁶ ergs cm⁻² sec⁻¹) inhibit the onset of mitosis; the cells remain in interphase instead. The inhibition of cell division is reversed after the end of the light treatment. Meiosis is not affected by light. Part of the premeiotic G2 phase is light sensitive. No arrest of DNA-, RNA- or protein synthesis is involved in the photoinhibition of onset of mitosis. Irradiation with blue light decreases the α- absorption band of cytochrome a₃ but not of cytochrome a. This effect on cytochrome oxidase, however, is observed in all stages of meitotic prophase as well as in G2 cells.     

Quantum Requirement for Photoreactivation of Inactivated Nitrate Reductase of Neurospora crassa Strain al-2, bd

Chemically inactivated nitrate reductase of Neurospora crassa strain al-2, bd can be photoreactivated by blue light. The quantum requirement for this reaction in the presence of exogenous FMN was determined with different light intensities. The results are discussed with the alternate assumptions that free FMN or photoactivated FAD bound to the nitrate reductase molecule is the reactivating species.     

Comparative analysis of uncoupling protein 4 distribution in various tissues under physiological conditions and during development

UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4's subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the protein's possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation.