Interference of CLN6 mutants

CLN6 (Ceroid Lipofuscinosis, Neuronal, 6) is a 311-amino acid protein spanning the endoplasmic reticulum membrane. Mutations in CLN6 are linked to CLN6 disease, a hereditary neurodegenerative disorder categorized into the neuronal ceroid lipofuscinoses. CLN6 disease is an autosomal recessive disorder and individuals affected with this disease have two identical (homozygous) or two distinct (compound heterozygous) CLN6 mutant alleles. Little has been known about CLN6’s physiological roles and the disease mechanism. We recently found that CLN6 prevents protein aggregate formation, pointing to impaired CLN6’s anti-aggregate activity as a cause for the disease. To comprehensively understand the pathomechanism, overall anti-aggregate activity derived from two different CLN6 mutants needs to be investigated, considering patients compound heterozygous for CLN6 alleles. We focused on mutant combinations involving the S132CfsX18 (132fsX) prematurely terminated protein, produced from the most frequent mutation in CLN6. The 132fsX mutant nullified anti-aggregate activity of the P299L CLN6 missense mutant but not of wild-type CLN6. Wild-type CLN6’s resistance to the 132fsX mutant was abolished by replacement of amino acids 297–301, including Pro297 and Pro299, with five alanine residues. Given that removal of CLN6’s C-terminal fifteen amino acids 297–311 (luminal tail) did not affect the resistance, we suggested that CLN6’s luminal tail, when unleashed from Pro297/299-mediated conformational constraints, is improperly positioned by the 132fsX mutant, thereby blocking the induction of anti- aggregate activity. We here reveal a novel mechanism for dissipating CLN6 mutants’ residual functions, providing an explanation for the compound heterozygosity-driven pathogenesis

Hymenobacter nivis sp nov., isolated from red snow in Antarctica

A novel aerobic bacterial strain, P3(T), was isolated from a red snow obtained from Antarctica. Cells of strain P3(T) were rod-shaped, non-motile, catalase-negative, oxidase-positive and Gramstain-negative. Growth was observed at temperatures ranging from 0 to 25 degrees C, with optimum growth at 15 degrees C. The pH range for growth was pH 5.3-7.8. The G+C content of the genomic DNA was 55.0 mol%. The major components in the fatty acid profile were iso-C-15: 0, summed feature 4 (iso-C-17:1 I and/or anteiso-C-17:1 B), anteiso-C-15:0 and summed feature 3 (isoC(16:1)omega 7c and/or iso-C(16:1 omega)6c). The predominant isoprenoid quinone was MK-7. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the novel isolate was a member of the genus Hymenobacter, and the strain showed highest sequence similarity (94 %) with Hymenpbacter glaciei VUG-A130(T), Hymenobacter soli PB17(T) and Hymenobacter antarcticus VUG-A42aa(T). On the basis of phylogenetic and phenotypic properties strain P3(T) represents a novel species of the genus Hymenobacter (for which the name Hymenobacter nivis sp. nov. is proposed. The types strain is P3(T) (= DSM 101755(T) = NBRC 111535(T))

Organellar DNA Polymerases in Complex Plastid-Bearing Algae

DNA replication in plastids and mitochondria is generally regulated by nucleus-encoded proteins. In plants and red algae, a nucleus-encoded enzyme called POP (plant and protist organellar DNA polymerase) is involved in DNA replication in both organelles by virtue of its dual localization. POPs are family A DNA polymerases, which include bacterial DNA polymerase I (PolI). POP homologs have been found in a wide range of eukaryotes, including plants, algae, and non-photosynthetic protists. However, the phylogeny and subcellular localizations of POPs remain unclear in many algae, especially in secondary and tertiary plastid-bearing groups. In this study, we report that chlorarachniophytes possess two evolutionarily distinct POPs, and fluorescent protein-tagging experiments demonstrate that they are targeted to the secondary plastids and mitochondria, respectively. The timing of DNA replication is different between the two organelles in the chlorarachniophyte Bigelowiella natans, and this seems to be correlated to the transcription of respective POP genes. Dinoflagellates also carry two distinct POP genes, possibly for their plastids and mitochondria, whereas haptophytes and ochrophytes have only one. Therefore, unlike plants, some algal groups are likely to have evolved multiple DNA polymerases for various organelles. This study provides a new insight into the evolution of organellar DNA replication in complex plastid-bearing organisms

Microglial re-modeling contributes to recovery from ischemic injury of rat brain: A study using a cytokine mixture containing granulocyte-macrophage colony-stimulating factor and interleukin-3

Ischemic stroke is a leading cause of mortality and permanent disability. Chronic stroke lesions increase gradually due to the secondary neuroinflammation that occurs following acute ischemic neuronal degeneration. In this study, the ameliorating effect of a cytokine mixture consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 was evaluated on ischemic brain injury using a rat stroke model prepared by transient middle cerebral artery occlusion (tMCAO). The mixture reduced infarct volume and ameliorated ischemia-induced motor and cognitive dysfunctions. Sorted microglia cells from the ischemic hemisphere of rats administered the mixture showed reduced mRNA expression of tumor necrosis factor (TNF)-α and IL-1β at 3 days post-reperfusion. On flow cytometric analysis, the expression of CD86, a marker of pro-inflammatory type microglia, was suppressed, and the expression of CD163, a marker of tissue-repairing type microglia, was increased by the cytokine treatment. Immunoblotting and immunohistochemistry data showed that the cytokines increased the expression of the anti-apoptotic protein Bcl-xL in neurons in the ischemic lesion. Thus, the present study demonstrated that cytokine treatment markedly suppressed neurodegeneration during the chronic phase in the rat stroke model. The neuroprotective effects may be mediated by phenotypic changes of microglia that presumably lead to increased expression of Bcl-xL in ischemic lesions, while enhancing neuronal survival

Tumor size and proliferative marker geminin levels associated with SUVmax levels on PET for breast cancers

It has been well established that maximum standardized uptake value (SUVmax) for 18F-fluorodeoxyglucose positron-emission tomography/computed tomography (FDG PET/CT) is clinically useful for evaluating treatment efficacy as well as predicting prognosis of breast cancer patients. Although SUVmax reflects increased glucose uptake and metabolism possibly induced by activation of growth factor signaling or TP53 dysfunction, tumor characteristics of SUVmax-high breast cancers remain to be elucidated. For the present study, we used immunohistochemical staining to investigate expressions of phospho-ribosomal protein S6 (pS6, downstream molecule of phosphatidyl inositol 3-kinase/Akt/mammalian target of the rapamycin/S6K pathway) and phosphor-p44/42 mitogen-activated protein kinase (pMAPK). Expression levels of TP53 and proliferative marker geminin as well as Ki67 were also examined by means of immunostaining in 163 invasive breast cancers. Cutoff values were set at 10% for pS6, 20% for pMAPK and TP53, and 4% for geminin. The SUVmax levels were significantly higher in the pS6-positive (p = 0.0173), TP53-positive (p = 0.0207) and geminin-high cancers (p2cm and geminin-high showed SUVmax-high, while only 6 of 49 (12.2%) breast cancers ≤2cm in size and with low geminin levels were SUVmax-high. In conclusion, we could determine that breast cancers with a large tumor and a geminin-high rather than Ki67- high proliferative marker were significantly associated with high levels of SUVmax. These findings may signify that SUVmax reflects tumor characteristics with high proliferative activity but not activation of mTOR/S6K and MAPK pathways or increased glucose metabolism due to dysfunction of TP53

Organellar DNA Polymerases in Complex Plastid-Bearing Algae

DNA replication in plastids and mitochondria is generally regulated by nucleus-encoded proteins. In plants and red algae, a nucleus-encoded enzyme called POP (plant and protist organellar DNA polymerase) is involved in DNA replication in both organelles by virtue of its dual localization. POPs are family A DNA polymerases, which include bacterial DNA polymerase I (PolI). POP homologs have been found in a wide range of eukaryotes, including plants, algae, and non-photosynthetic protists. However, the phylogeny and subcellular localizations of POPs remain unclear in many algae, especially in secondary and tertiary plastid-bearing groups. In this study, we report that chlorarachniophytes possess two evolutionarily distinct POPs, and fluorescent protein-tagging experiments demonstrate that they are targeted to the secondary plastids and mitochondria, respectively. The timing of DNA replication is different between the two organelles in the chlorarachniophyte Bigelowiella natans, and this seems to be correlated to the transcription of respective POP genes. Dinoflagellates also carry two distinct POP genes, possibly for their plastids and mitochondria, whereas haptophytes and ochrophytes have only one. Therefore, unlike plants, some algal groups are likely to have evolved multiple DNA polymerases for various organelles. This study provides a new insight into the evolution of organellar DNA replication in complex plastid-bearing organisms