Xenobiotics exposure increases endoplasmic reticulum (ER) proliferation and cytochrome P-450 (CYP) induction to sustain metabolic requirements. Whether autophagy is essential for the removal of ...excess ER and CYP and whether an autophagy receptor is involved in this process in mammals remains elusive. In this study, we show that autophagy is induced in mouse livers after withdrawal of the hepatic mitogen 1,4-bis2-(3,5-dichloropyridyloxy) benzene (TCPOBOP). Although isolated autophagosomes, autolysosomes, and lysosomes from mouse livers after withdrawal of TCPOBOP contained ER proteins, those in control mouse livers did not. Liver-specific Atg5 knockout mice had higher basal hepatic ER content that was further increased and sustained after withdrawal of TCPOBOP compared with wild-type mice. In addition to regulating ER degradation, our results also suggest that autophagy plays a role in regulating the homeostasis of hepatic CYP because blocking autophagy led to increased CYP2B10 accumulation either at the basal level or following TCPOBOP withdrawal. Furthermore, we found that the autophagy receptor protein sequestosome 1 (SQSTM1)/p62 is associated with the ER. After withdrawal of TCPOBOP, p62 knockout mice had increased ER content in the liver compared with wild-type mice. These results suggest that p62 may act as an autophagy receptor for the autophagic removal of excess ER in the mouse liver. Taken together, our results indicate that autophagy is important for the removal of excess ER and hepatic CYP enzymes in mouse livers, a process associated with the autophagy receptor protein p62.
Protected areas are cornerstones of biodiversity conservation, but they are in danger of becoming islands in a sea of human dominated landscapes. Our question was if protected areas may even foster ...development in their surroundings because they provide amenities that attract development, thus causing the isolation of the ecosystems they were designed to protect. Our study analyzed historic aerial photographs and topographical maps to reconstruct road development and building growth within and around Indiana Dunes and Pictured Rocks National Lakeshores in the U.S. Great Lakes region from 1938 to 2005, and to estimate the effects of park creation in 1966 on changes in landscape patterns. Historic U.S. census housing density data were used as a baseline to compare observed changes to. Our results showed that park establishment was effective in reducing and stopping the fragmenting impact of development within park boundaries. However, increased amenity levels following park establishment led to enhanced development in the surroundings of both parks. In the extreme case of Indiana Dunes, building density outside the park increased from 45 to 200
buildings/km
2 and road density almost doubled from 3.6 to 6.6
km/km
2 from 1938 to 2005. Development rates of change were much higher than in the broader landscape, particularly after park establishment. The potential amenity effect was up to 9500 new buildings in the 3.2-km zone around Indiana Dunes between 1966 and 2005. For Pictured Rocks the absolute effect was smaller but up to 70% of the observed building growth was potentially due to amenity effects. Our findings highlight the need for conservation planning at broader scales, incorporating areas beyond the boundaries of protected areas.
The wobbler mouse (phenotype WR; genotype wr/wr) has been investigated as a model for neurodegenerative diseases like SMA and ALS. A new diagnostic marker based on a polymorphism in the closely ...linked chaperonine gene Cct4 enabled us to diagnose the allelic status at the wr locus within the original background strain C57BL/6. Using this marker, we investigated the spatiotemporal progression of neuropathology in WR mice from postnatal day (d.p.n.) 10 to 60. Neurodegeneration starts at 13 d.p.n. in the thalamus (N. ventralis), in deep cerebellar nuclei, brain stem (N. vestibularis) and spinal cord interneurons. The motor nuclei of spinal nerves and motoneurons degenerate from 15 d.p.n. onward. Reactive astrocytes are observed around 17 d.p.n. in the white and grey matter of the spinal cord. Microgliosis occurs only from 23 d.p.n. onward. Our data demonstrate that in the WR disease, neurodegeneration in thalamus, cerebellum, and brain stem precedes motoneuron degeneration, astrogliosis and microgliosis.
The autosomal recessive mutation wobbler of the mouse (phenotype WR; genotype wr/wr) causes muscular atrophy due to motoneuron degeneration with 100% penetrance on the standard Mus musculus ...laboratorius C57BL/6J background. In inter- and backcrosses with M. m. castaneus strain CAST/EI we have observed a variability in the severity of neurological symptoms. Approximately 15% of the WR (wr/wr) CAST/B6 hybrids were modified wobbler (WR*) mice defined by an aggravated neuromuscular phenotype with hindlimbs severely affected in addition to forelimbs. Histologically the overt WR* phenotype was paralleled by a caudally extended neurodegeneration in the ventral horn of the spinal cord with severe astrogliosis, and levels of acetylcholine receptor alpha-subunit mRNA in leg muscle much higher than in standard WR mice. Segregation analysis, using 68 polymorphic autosomal markers in a whole genome scan, revealed a major modifier gene locus, termed wrmod1, on chromosome 14. Individual recombination events in chromosome 14 consomic mice narrowed the wrmod1 candidate region to a 29 cM interval between D14MIT154 and D14MIT105, a region homologous to human chromosome 13q. Our analysis provides access to genes that modify neurodegeneration, the human counterparts of which may be responsible for the variable expression of hereditary spinal muscular atrophies.
Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777. We have ...studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC
50 for nNOS was 18±6 μM GST-PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome
c reductase activity, the
K
M for
l-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.
The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mutant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexpression system. The purified protein was soluble and highly ...active (0.2-0.7 micromol of l-citrulline. mg-1.min-1), contained 0. 77+/-0.01 equivalent of haem per subunit, showed a Soret maximum at 396 nm, and exhibited only minor uncoupling of NADPH oxidation in the absence of l-arginine or tetrahydrobiopterin. Radioligand binding studies revealed KD values of 147+/-24.1 nM and 52+/-9.2 nM for specific binding of tetrahydrobiopterin in the absence and presence of 0.1 mM l-arginine respectively. The positive co-operative effect of l-arginine was due to a pronounced decrease in the rate of tetrahydrobiopterin dissociation (from 1.6+/-0.5 to 0. 3+/-0.1 min-1). Low-temperature SDS gel electrophoresis showed that approx. 80% of the protein migrated as haem-containing dimer after preincubation with l-arginine and tetrahydrobiopterin. Gel-filtration chromatography yielded one peak with a Stokes radius of 6.8+/-0.04 nm, corresponding to a hydrodynamic volume of 1. 32x10(-24) m3, whereas haem-deficient preparations (approx. 0.3 equivalent per subunit) contained an additional protein species with a hydrodynamic radius of 5.1+/-0.2 nm and a corresponding volume of 0.55x10(-24) m3, suggesting that haem availability regulates eNOS dimerization.
Metrifonate, a long-acting cholinesterase (ChE) inhibitor with very low toxicity in warm-blooded animals, inhibits rat brain and serum cholinesterase (ChE) in vitro through its hydrolytic degradation ...product, dichlorvos. This conclusion is based on the finding that metrifonate-induced ChE inhibition showed the same pH dependence as its reported dehydrochlorination to dichlorvos. The ChE inhibition induced by dichlorvos was not pH dependent. It was mediated by a competitive drug interaction with the catalytic site of the enzyme, which led to irreversible inhibition within several minutes of incubation. After this time, addition of further substrate to the inhibited enzyme was not able to promote drug dissociation and hence enzyme reactivation. Similar characteristics of inhibition, i.e. interaction with the substrate binding site and time-dependent switch to non-competitive inhibition were observed with the reference compound, physostigmine. However, the physostigmine-induced inhibition of ChE could be readily reversed by further substrate addition. Another reference compound, tetrahydroaminoacridine (THA), also induced a reversible inhibition of rat brain and serum cholinesterase, but with a mechanism of action different from that of both dichlorvos and physostigmine in that enzyme inhibition occurred rapidly upon drug addition at an allosteric site on the enzyme surface. It is suggested that the unique slow release plus the slow inhibition of ChE by dichlorvos is responsible for the lower toxicity of metrifonate compared to that of directly acting ChE inhibitors.
Two sample preparation techniques (ultrafine powder pellet and flux‐free fusion glass) for LA‐ICP‐MS bulk analysis of granitoids were compared. Ultrafine powder particles produced by wet milling were ...characterised as d50: ~ 1.0 μm, d90: ~ 5.0 μm. Agate abrasion (balls and vial) during wet milling affects only SiO2 measurements and is negligible for other elements. For the flux‐free fusion glass, a second grinding of the initial fused glass and re‐melting were necessary to produce compositionally homogeneous glasses. Nickel and Cu can be contaminated during the fusion procedure, and Sn and Pb were depleted after the melting process. The homogeneity of fusion glasses was comparable to that of MPI‐DING glasses, while the powder pellets were less homogeneous. This heterogeneity is ascribed to large (up to 10 μm) crystal fragments (e.g., biotite) persisting in powders even after 45 min of milling. For most elements of interest, both preparation techniques give reliable LA‐ICP‐MS results of granitoid reference materials within 10% of the reference values. Thus, we can recommend both techniques to avoid common problems associated with acid dissolution ICP‐MS. For high‐precision measurements (especially Zr, Hf, Th and U), the flux‐free fusion glass technique is a better choice than ultrafine powder pellets.
Key Points
Ultrafine powder particles produced by wet milling were characterised as d50 ∼1.0 μm, d90 ∼5.0 μm.
For the flux‐free fusion glass, a second grinding of the initial fused glass and re‐melting was necessary to produce compositionally homogeneous glasses.
Homogeneity of fusion glasses was comparable to that of MPI‐DING glasses, while the powder pellets were less homogeneous.
Electrons bound in highly charged heavy ions such as hydrogen-like bismuth
Bi
experience electromagnetic fields that are a million times stronger than in light atoms. Measuring the wavelength of ...light emitted and absorbed by these ions is therefore a sensitive testing ground for quantum electrodynamical (QED) effects and especially the electron-nucleus interaction under such extreme conditions. However, insufficient knowledge of the nuclear structure has prevented a rigorous test of strong-field QED. Here we present a measurement of the so-called specific difference between the hyperfine splittings in hydrogen-like and lithium-like bismuth
Bi
with a precision that is improved by more than an order of magnitude. Even though this quantity is believed to be largely insensitive to nuclear structure and therefore the most decisive test of QED in the strong magnetic field regime, we find a 7-σ discrepancy compared with the theoretical prediction.
During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic‐specific genes and subsequently develops into the first lymphatic structures, ...collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination‐based ultramicroscopy to entire wholemount‐immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF‐C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development.
Ultramicroscopy of wholemount mouse embryos uncovers the first, previously unknown lymphatic structures in mammals: the dorsal longitudinal lymphatic vessel and the ventral primordial thoracic duct, which eventually connect with the cardinal vein as previously described.
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