Neurofibrillary Tangles and the Deposition of a Beta Amyloid Peptide with a Novel N-Terminal Epitope in the Brains of Wild Tsushima Leopard Cats

Beta amyloid (Aβ) deposits are seen in aged individuals in many of the mammalian species that possess the same Aβ amino acid sequence as humans. Conversely, neurofibrillary tangles (NFT), the other hallmark lesion of Alzheimer’s disease (AD), are extremely rare in these animals. We detected Aβ deposits in the brains of Tsushima leopard cats (Prionailurus bengalensis euptilurus) that live exclusively on Tsushima Island, Japan. Aβ42 was deposited in a granular pattern in the neuropil of the pyramidal cell layer, but did not form argyrophilic senile plaques. These Aβ deposits were not immunolabeled with antibodies to the N-terminal of human Aβ. Sequence analysis of the amyloid precursor protein revealed an amino acid substitution at the 7th residue of the Aβ peptide. In a comparison with other mammalian animals that do develop argyrophilic senile plaques, we concluded that the alternative Aβ amino acid sequence displayed by leopard cats is likely to be related to its distinctive deposition pattern. Interestingly, most of the animals with these Aβ deposits also developed NFTs. The distributions of hyperphosphorylated tau-positive cells and the two major isoforms of aggregated tau proteins were quite similar to those seen in Alzheimer’s disease. In addition, the unphosphorylated form of GSK-3β colocalized with hyperphosphorylated tau within the affected neurons. In conclusion, this animal species develops AD-type NFTs without argyrophilic senile plaques.     

Genetic and mass spectrometry analyses of the unusual type IV-like pili of the archaeon Methanococcus maripaludis

The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (ΔflaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation.     

Improved production of L-lysine by disruption of stationary phase-specific rmf gene in Escherichia coli

Growth and rate, at which fermentation products are formed in cells, generally decreases during the stationary phase as a result of changes in gene expression. We focused on the rmf gene, which encodes the ribosome modulation factor protein, as a target for strain modification in order to improve the rate of L-lysine production in Escherichia coli. Increased expression of the rmf gene during the stationary phase was confirmed under various cultivation conditions using DNA macroarray analysis. Mutants with disrupted rmf were then generated from an L-lysine-producing E. coli strain. The rates of L-lysine accumulation and production were significantly increased in disruptants that were cultivated with excess phosphate. By contrast, a higher biomass was generated in disruptants that were grown under limited phosphate conditions. These results demonstrate that disruption of the rmf gene significantly affects L-lysine production and growth in E. coli.     

Genetic variation and gene flow of broadcast spawning and planula brooding coral,Goniastrea aspera (Scleractinia) in the Ryukyu Archipelago,southern Japan

The scleractinian coral Goniastrea aspera (Verrill) undergoes both broadcast spawning and planulae brooding in the Ryukyu Archipelago of southern Japan. Genetic variation and gene flow in G. aspera were studied using allozyme electrophoresis. We tested the hypothesis that gene flow is determined by the competency period of the planulae. We also assessed the relative contributions of sexual and asexual reproduction to recruitment. For the five staining systems surveyed, G. aspera encoded five polymorphic loci and one monomorphic locus. The genotype frequencies in each population significantly differed from the expected Hardy-Weinberg equilibrium (HWE), indicating that the local populations of G. aspera are not fully panmictic. The high ratio of the observed number of genotypes to the number of individuals (0.90 +/- 0.07, mean NG:N +/- SD) and the observed to expected genotypic diversity (0.84 +/- 0.11, mean GO:GE +/- SD) suggested that each population is likely maintained by sexual reproduction. The genetic differentiation (FST) and value of average number of migrants per generation (Nem) among and within regions ranged from 0.025 to 0.104 and 2.2 to 9.6, respectively. Comparisons with other species demonstrated that larva survival rates also influence gene flow. In addition, gene flow on distant reefs by planulae originating from spawning might prevent divergence by planulae originating from brooding for short-distant dispersal among and within populations of G. aspera in the Ryukyu Archipelago.     

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (∼ 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A₁ instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.     

Accumulation of calcium in human common iliac artery, aortic valve, xiphoid process, costal cartilage, posterior longitudinal ligament, trigeminal nerve, and rib accompanied by increase of magnesium

To examine whether an accumulation of Ca in the tissues was accompanied by an increase of Mg, the authros investigated the relationships between Ca and Mg contents in the common iliac arteries, aortic valves, xiphoid processes, costal cartilages, posterior longitudinal ligaments, trigeminal nerves, and ribs by inductively coupled plasma-atomic emission spectrometry. After the ordinary dissections by medical students were finished, the common iliac arteries, aortic valves, xiphoid processes, bilateral the fourth costal cartilages, posterior longitudinal ligaments between the fourth and fifth cervical vertebrae, trigeminal nerves, and bilateral the sixth ribs were resected from the subjects and elements were determined. It was found that there were extremely significant direct correlations between Ca and Mg contents in all of the common iliac arteries, aortic valves, costal cartilages, posterior longitudinal ligaments, and trigeminal nerves, whereas there were significant direct correlations in both the xiphoid processes and ribs. As for the tissues containing Ca higher than 20 mg/g, the average mass ratios of Mg/Ca were similar among the seven tissues. As Ca increased in all of the common iliac arteries, aortic valves, xiphoid processes, costal cartilages, posterior longitudinal ligaments, trigeminal nerves, and ribs, Mg increased simultaneously in the seven tissues.     

The benefits of being dominant: health correlates of male social rank and age in a marmot

The benefits of dominance may not come without costs, particularly for males. For example, the “immunocompetence handicap hypothesis” states that males with enhanced mating success allocate resources to enhance reproductive output at a cost to their current health, whereas the “resource quality hypothesis” predicts that high-ranking males may benefit from increased reproduction and good health. Whereas the predictions from each have been well tested in captive animals and in a variety of highly social primates, fewer studies have been carried out in free-living, facultatively social animals. Using adult male yellow-bellied marmots (Marmota flaviventer), we evaluated predictions of these hypotheses by examining the relationship between social rank and 2 health indicators—fecal glucocorticoid metabolite (FCM) levels, and neutrophil/lymphocyte (N/L) ratios—after accounting for variation explained by age, body mass, and seasonality. We found that higher-ranking males tended to have a lower N/L ratio (reflecting good health) than lower-ranking individuals, whereas FCM levels were not significantly related to rank. In addition, heavier male marmots had lower N/L ratios, whereas body mass was not associated with FCM levels. We also found that older adult males had lower FCM levels (reflecting less physiological stress) but higher N/L ratios than younger adults. Finally, we found that FCM levels decreased as the active season progressed and FCM levels were associated with the time of the day. Overall, our results suggest that socially-dominant male marmots enjoyed better, not worse health in terms of lower N/L ratios.     

Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens

Background

Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection.

Methods

RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006–2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems.

Results

rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%.

Conclusions

Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

     

Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana

Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO₂ concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.