Molecular cloning and gene expression of endoplasmic reticulum stress proteins in Japanese monkey, Macaca fuscata

BACKGROUND: Immunoglobulin heavy-chain binding protein (BiP), calreticulin (Crt), and protein disulfide isomerase (PDI), are major resident endoplasmic reticulum (ER) stress proteins which are involved in diverse roles relating to successful folding, assembly, intracellular localization, and degradation of other proteins. METHODS: In this study, we molecular cloned cDNAs for BiP, Crt, and PDI from Japanese monkey (Macaca fuscata), and analyzed tissue-specific expression of respective genes. RESULTS AND CONCLUSIONS: The lengths of protein-coding regions of these cDNAs for BiP, Crt and PDI are 1965, 1254, and 1533 bp, respectively. Each protein has a signal peptide and a KDEL motif in N- and C-terminal parts respectively, showing its intracellular localization to be the lumen of the ER. These stress proteins are highly conserved, showing that their similarities among mammals are more than 90% in the level of amino acid. The expression of the genes for stress proteins differed among the monkey tissues examined. BiP and PDI gene expression was predominant in secretory tissues such as liver and kidney, and brain tissues. But Crt gene expressed rather ubiquitously in a variety of tissues.     

Prevalence of a non-male-killing spiroplasma in natural populations of Drosophila hydei

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.     

Imprints of glacial refugia in the modern genetic diversity of Pinus sylvestris

Aim To understand the impact of glacial refugia and migration pathways on the modern genetic diversity of Pinus sylvestris. Location The study was carried out throughout Europe. Methods An extended set of data of pollen and macrofossil remains was used to locate the glacial refugia and reconstruct the migrating routes of P. sylvestris throughout Europe. A vegetation model was used to simulate the extent of the potential refugia during the last glacial period. At the same time a genetic survey was carried out on this species. Results The simulated distribution of P. sylvestris during the last glacial period is coherent with the observed fossil data, which showed a patchy distribution of the refugia between c. 40° N and 50° N. Several migrational fronts were detected within the Iberian and the Italian peninsulas, and outside the Hungarian plain and around the Alps. The modern mitochondrial DNA depicted three different haplotypes for P. sylvestris. Two distinct haplotypes were restricted to northern Spain and Italy, and the third haplotype dominated most of the present‐day remaining distribution range of P. sylvestris in Europe. Main conclusions During the last glacial period P. sylvestris was constrained under severe climatic conditions to survive in scattered and restricted refugial areas. Combining palaeoenvironmental data, vegetation modelling and the genetic data, we have shown that the long‐term isolation in the glacial refugia and the migrational process during the Holocene have played a major role in shaping the modern genetic diversity of P. sylvestris in Europe.     

Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt

Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine–synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.     

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.     

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.     

Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese

Background

To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated.

Methods and Principal Findings

We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10⁻¹⁰). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients.

Conclusions and Significance

In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.     

Binding Pocket Optimization by Computational Protein Design

Engineering specific interactions between proteins and small molecules is extremely useful for biological studies, as these interactions are essential for molecular recognition. Furthermore, many biotechnological applications are made possible by such an engineering approach, ranging from biosensors to the design of custom enzyme catalysts. Here, we present a novel method for the computational design of protein-small ligand binding named PocketOptimizer. The program can be used to modify protein binding pocket residues to improve or establish binding of a small molecule. It is a modular pipeline based on a number of customizable molecular modeling tools to predict mutations that alter the affinity of a target protein to its ligand. At its heart it uses a receptor-ligand scoring function to estimate the binding free energy between protein and ligand. We compiled a benchmark set that we used to systematically assess the performance of our method. It consists of proteins for which mutational variants with different binding affinities for their ligands and experimentally determined structures exist. Within this test set PocketOptimizer correctly predicts the mutant with the higher affinity in about 69% of the cases. A detailed analysis of the results reveals that the strengths of PocketOptimizer lie in the correct introduction of stabilizing hydrogen bonds to the ligand, as well as in the improved geometric complemetarity between ligand and binding pocket. Apart from the novel method for binding pocket design we also introduce a much needed benchmark data set for the comparison of affinities of mutant binding pockets, and that we use to asses programs for in silico design of ligand binding.     

Neanderthal Extinction by Competitive Exclusion

Background

Despite a long history of investigation, considerable debate revolves around whether Neanderthals became extinct because of climate change or competition with anatomically modern humans (AMH).

Methodology/Principal Findings

We apply a new methodology integrating archaeological and chronological data with high-resolution paleoclimatic simulations to define eco-cultural niches associated with Neanderthal and AMH adaptive systems during alternating cold and mild phases of Marine Isotope Stage 3. Our results indicate that Neanderthals and AMH exploited similar niches, and may have continued to do so in the absence of contact.

Conclusions/Significance

The southerly contraction of Neanderthal range in southwestern Europe during Greenland Interstadial 8 was not due to climate change or a change in adaptation, but rather concurrent AMH geographic expansion appears to have produced competition that led to Neanderthal extinction.