I-κBα depletion by transglutaminase 2 and μ-calpain occurs in parallel with the ubiquitin-proteasome pathway

Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-κB through the polymerization-mediated depletion of I-κBα without IKK activation. This NF-κB activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-κBα, which raises the question of how the polymerized I-κBα can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-κBα polymers in MCF7 cells transfected with TGase 2, and induced high levels of I-κBα polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-κBα polymers in HEK-293 cells in case of TGase 2 transfection either with I-κBα or I-κBα mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-κBα polymers and I-κBα, concurrent with an inhibition of NF-κB activity in MDA-MB-231 cells. This suggests that μ-calpain proteasome-dependent I-κBα polymer degradation may contribute to cancer progression through constitutive NF-κB activation.     

Atmin modulates Pkhd1 expression and may mediate Autosomal Recessive Polycystic Kidney Disease (ARPKD) through altered non-canonical Wnt/Planar Cell Polarity (PCP) signalling

Autosomal Recessive Polycystic Kidney Disease (ARPKD) is a genetic disorder with an incidence of ~1:20,000 that manifests in a wide range of renal and liver disease severity in human patients and can lead to perinatal mortality. ARPKD is caused by mutations in PKHD1, which encodes the large membrane protein, Fibrocystin, required for normal branching morphogenesis of the ureteric bud during embryonic renal development. The variation in ARPKD phenotype suggests that in addition to PKHD1 mutations, other genes may play a role, acting as modifiers of disease severity. One such pathway involves non-canonical Wnt/Planar Cell Polarity (PCP) signalling that has been associated with other cystic kidney diseases, but has not been investigated in ARPKD. Analysis of the Atmin^Gpg6 mouse showed kidney, liver and lung abnormalities, suggesting it as a novel mouse tool for the study of ARPKD. Further, modulation of Atmin affected Pkhd1 mRNA levels, altered non-canonical Wnt/PCP signalling and impacted cellular proliferation and adhesion, although Atmin does not bind directly to the C-terminus of Fibrocystin. Differences in ATMIN and VANGL2 expression were observed between normal human paediatric kidneys and age-matched ARPKD kidneys. Significant increases in ATMIN, WNT5A, VANGL2 and SCRIBBLE were seen in human ARPKD versus normal kidneys; no substantial differences were seen in DAAM2 or NPHP2. A striking increase in E-cadherin was also detected in ARPKD kidneys. This work indicates a novel role for non-canonical Wnt/PCP signalling in ARPKD and suggests ATMIN as a modulator of PKHD1.     

Movements of beluga whales (Delphinapterus leucas) in Bristol Bay,Alaska

We describe the annual distribution of beluga whales (Delphinapterus leucas) in Bristol Bay, Alaska, using data from 31 satellite‐linked transmitters during 2002–2011. Bristol Bay has one of the largest and best studied Pacific salmon (Oncorhynchus spp.) fisheries in the world, allowing us to link the seasonal distribution of belugas to that of salmon. During salmon migrations, beluga movements were restricted to river entrances. Belugas generally did not relocate to different river entrances or change bays during peak salmon periods. However, the location of belugas was not related to the number of salmon passing counting towers, suggesting that belugas were either selecting locations that were good for catching salmon or there were simply more salmon than belugas needed to supply their nutritional needs. The distribution of belugas expanded after salmon runs ended, and was greatest in winter when belugas ranged beyond the inner bays, traveling as far west as Cape Constantine. Belugas continued to frequent the inner bays in winter whenever sea ice conditions allowed, e.g., when winds moved sea ice offshore; however, they were never located south of the southern ice edge in open water or outside of Bristol Bay.     

Replication and fine mapping of asthma-associated loci in individuals of African ancestry

Asthma originates from genetic and environmental factors with about half the risk of disease attributable to heritable causes. Genome-wide association studies, mostly in populations of European ancestry, have identified numerous asthma-associated single nucleotide polymorphisms (SNPs). Studies in populations with diverse ancestries allow both for identification of robust associations that replicate across ethnic groups and for improved resolution of associated loci due to different patterns of linkage disequilibrium between ethnic groups. Here we report on an analysis of 745 African-American subjects with asthma and 3,238 African-American control subjects from the Candidate Gene Association Resource (CARe) Consortium, including analysis of SNPs imputed using 1,000 Genomes reference panels and adjustment for local ancestry. We show strong evidence that variation near RAD50/IL13, implicated in studies of European ancestry individuals, replicates in individuals largely of African ancestry. Fine mapping in African ancestry populations also refined the variants of interest for this association. We also provide strong or nominal evidence of replication at loci near ORMDL3/GSDMB, IL1RL1/IL18R1, and 10p14, all previously associated with asthma in European or Japanese populations, but not at the PYHIN1 locus previously reported in studies of African-American samples. These results improve the understanding of asthma genetics and further demonstrate the utility of genetic studies in populations other than those of largely European ancestry.     

Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry

Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel.     

Structural and functional characterization of Streptococcus pyogenes Cas2 protein under different pH conditions

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0–9.0). Although S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes.     

Nicotinic Receptor-Mediated Release of Noradrenaline in the Human Neuroblastoma SH-SY5Y

Abstract: Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12- O -tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 μ M mecamylamine but not by 1 μ M atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 μ M Bay K 8644 (an L-type calcium agonist) and inhibited by 1 μ M nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 μ M desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.     

Defective ion regulation in a class of membrane-excitation mutants in Paramecium

The “paranoiac” mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na⁺, unlike wild-type paramecia, which jerk back and forth in Na⁺ solutions. The paranoiac mutants in Na⁺ solutions also show large losses of cellular K⁺ and large influxes of Na⁺. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na⁺-dependent loss of cellular K⁺ and a much smaller Na⁺ influx. In K⁺-containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K⁺ content.The Na⁺ influx, the K⁺ loss, and the duration of backward swimming are all proportional to the extracellular Na⁺ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na⁺ influxes and the large K⁺ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.     

Composition of cellulin,the unique chitin-glucan granules of the fungus,Apodachlya sp.

Cellulin granules, the polysaccharide inclusions found uniquely in oomycetous fungi of the order Leptomitales, were isolated from Apodachlya sp. The granules were prepared free of cell wall and cytoplasmic contaminants. Biochemical analyses and X-ray diffraction studies demonstrated that the granules were composed of 60% chitin and 39% glucan consisting of β-1,3-and β-1,6-linked glucose units. A protein content of only 0.1% was attributed to an insignificant amount of cytoplasmic contamination. Isolated granules and those in situ showed no apparent differences in their microscopic form.