The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

Geno viewer, a SAM/BAM viewer tool

The ever evolving Next Generation Sequencing technology is calling for new and innovative ways of data processing and visualization. Following a detailed survey of the current needs of researchers and service providers, the authors have developed GenoViewer: a highly user-friendly, easy-to-operate SAM/BAM viewer and aligner tool. GenoViewer enables fast and efficient NGS assembly browsing, analysis and read mapping. It is highly customized, making it suitable for a wide range of NGS related tasks. Due to its relatively simple architecture, it is easy to add specialised visualization functionalities, facilitating further customised data analysis. The software's source code is freely available; it is open for project and task-specific modifications. AVAILABILITY: The database is available for free at http://www.genoviewer.com/     

Partial 7q Isochromosome in Bone Marrow Following Treatment of Hodgkin's Disease

We report on a 29-year-old female followed for relapsed Hodgkin's disease. She had been diagnosed with Hodgkin's disease at 20 years of age and had been treated with chemotherapy. She had been in remission for six years when she relapsed, at which time she received chemotherapy for bone marrow transplant (BMT). After failure of BMT, she received additional chemotherapies with growth factors and radiation treatment. A bone marrow biopsy showed moderate hypercellularity with erythroid hyperplasia, but the karyotype had an abnormal clone containing an isochromosome derived from a 7q22 deletion.     

Recruitment of histone modifications by USF proteins at a vertebrate barrier element

The chicken beta-globin 5'HS4 insulator element acts as a barrier to the encroachment of chromosomal silencing. Endogenous 5'HS4 sequences are highly enriched with histone acetylation and H3K4 methylation regardless of neighboring gene expression. We report here that 5'HS4 elements recruit these histone modifications when protecting a reporter transgene from chromosomal silencing. Deletion studies identified a single protein binding site within 5'HS4, footprint IV, that is necessary for the recruitment of histone modifications and for barrier activity. We have determined that USF proteins bind to footprint IV. USF1 is present in complexes with histone modifying enzymes in cell extracts, and these enzymes specifically interact with the endogenous 5'HS4 element. Knockdown of USF1 expression leads to a loss of histone modification recruitment and subsequent encroachment of H3K9 methylation. We propose that barrier activity requires the constitutive recruitment of H3K4 methylation and histone acetylation at multiple residues to counteract the propagation of condensed chromatin structures.     

Elevation as a barrier: genetic structure for an Atlantic rain forest tree (Bathysa australis) in the Serra do Mar mountain range,SE Brazil

Distance and discrete geographic barriers play a role in isolating populations, as seed and pollen dispersal become limited. Nearby populations without any geographic barrier between them may also suffer from ecological isolation driven by habitat heterogeneity, which may promote divergence by local adaptation and drift. Likewise, elevation gradients may influence the genetic structure and diversity of populations, particularly those marginally distributed. Bathysa australis (Rubiaceae) is a widespread tree along the elevation gradient of the Serra do Mar, SE Brazil. This self‐compatible species is pollinated by bees and wasps and has autochoric seeds, suggesting restricted gene dispersal. We investigated the distribution of genetic diversity in six B. australis populations at two extreme sites along an elevation gradient: a lowland site (80–216 m) and an upland site (1010–1100 m.a.s.l.). Nine microsatellite loci were used to test for genetic structure and to verify differences in genetic diversity between sites. We found a marked genetic structure on a scale as small as 6 km (F_ST = 0.21), and two distinct clusters were identified, each corresponding to a site. Although B. australis is continuously distributed along the elevation gradient, we have not observed a gene flow between the extreme populations. This might be related to B. australis biological features and creates a potential scenario for adaptation to the different conditions imposed by the elevation gradient. We failed to find an isolation‐by‐distance pattern; although on the fine scale, all populations showed spatial autocorrelation until ~10‐20 m. Elevation difference was a relevant factor though, but we need further sampling effort to check its correlation with genetic distance. The lowland populations had a higher allelic richness and showed higher rare allele counts than the upland ones. The upland site may be more selective, eliminating rare alleles, as we did not find any evidence for bottleneck.     

Structure and synthesis of malformin A1

The structure of malformin A₁, a metabolic product of Aspergillus niger, was reexamined and the sequence of its amino acid constituents established as

The cyclopentapeptide-disulfide corresponding to this structure was prepared through stepwise synthesis of the protected pentapeptide derivative, benzyloxy-carbonyl-l-isoleucyl-S-benzyl-d-cysteinyl-S-benzyl-d-cysteinyl-l-valyl-d-leucine methylester, which in turn was converted to the hydrazide, partially deprotected, and cyclized via the azide. On removal of the S-benzyl groups and oxidation to the disulfide, a synthetic material was obtained that was indistinguishable from natural malformin A₁ and was as equally potent in causing curvatures on corn roots.     

Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.

Methodology/Principal Findings

Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.

Conclusion/Significance

Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.     

Synthetic substrates for measuring activity of autophagy proteases: Autophagins (Atg4)

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A₂ (LC3B-PLA₂), which upon cleavage releases active PLA₂ for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA₂ substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA₂ substrate afforded substantially higher catalytic rates (k_cat/K_m 5.26 × 10⁵ M⁻¹/sec⁻¹) than Ac-GTFG-AFC peptide (0.92 M⁻¹/sec⁻¹), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.Key words: autophagin, fluorogenic assay, tetrapeptide, phospholipase A2, LC3     

Division-site selection, cell separation, and formation of anucleate minicells inSchizosaccharomyces pombe mutants resistant to cell-wall lytic enzymes

Summary In most eukaryotic organisms that have cell walls, cell separation or cytokinesis is a degradative enzymatic process. In the fission yeastSchizosaccharomyces pombe, it is a post-M-phase event that includes the degradation of part of the cell wall and the primary septum. We describe the isolation of mutants partially defective in cytokinesis by enrichment of clones resistant to cell-wall lytic enzymes. The mutations confer mycelial morphology (chains of non-separated cells) and define four genes.Sep2-SA2 was subjected to detailed genetic and cytological analysis. Its cells frequently form complex septa composed of multiple layers, which appear as twin septa separated by anucleate minicells if the cell length is extended. This suggests that a polar signal-like mechanism may also operate inS. pombe during division-site selection andsep2 ⁺ takes part in it.Sep2 ⁺ seems to be involved in several cell cycle functions because its mutation can transiently block cell-cycle progression after nuclear division and provoke a transition from haploidy to diploidy in the double mutantsep2-SA2 cexl-SA2. Cexl-SA2 is another novel mutation which causes cell-length extension.Abbreviations DAPI 4,6-diamidino-2-phenylindole - YEA yeast extract agar - YEL yeast extract liquid - SMA synthetic minimal agar - MEA malt extract agar