Does dietary arsenic and mercury affect cutaneous bleeding time and bloods lipids in humans

The online version of the original article can be found at     

Circadian and Circannual Variations of Cell Cycle Distribution in the Mouse Bone Marrow

The cell cycle distribution of bone marrow cells from the femurs of female C3H mice has been investigated by flow cytometry according to the time of the day and month of the year. Both circadian and seasonal variations were found for the different cell cycle phases as well as the total cell numbers per femur. Both the mesor, the acrophase and the amplitude of the S, G₂ and (G₁ + G₀) phases varied significantly in some months, while in other months only insignificant rhythms were found. The relative cell cycle distribution only partly reflected variations in the total numbers of proliferating cells, since the total cell number per femur was also variable.

The total numbers of cells in DNA synthesis seem to be higher in the first part of the year, indicating increased cell proliferation during winter and spring. In this period the acrophases of DNA synthesis and G₂ were in the morning, while the second half of the year showed the peak later in the day.

In general, hemopoietic cell proliferation seems to constitute a labile equilibrium with rapidly changing activities.     

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine Evidence for different pathways

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [³H]retinol and ¹⁴C-labelled vitamin D-3, ¹⁴C-labeled vitamin D-3 appeared later in the intestinal lymph than [³H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.     

Determinants of parasitoid communities of willow‐galling sawflies: habitat overrides physiology,host plant and space

Studies on the determinants of plant–herbivore and herbivore–parasitoid associations provide important insights into the origin and maintenance of global and local species richness. If parasitoids are specialists on herbivore niches rather than on herbivore taxa, then alternating escape of herbivores into novel niches and delayed resource tracking by parasitoids could fuel diversification at both trophic levels. We used DNA barcoding to identify parasitoids that attack larvae of seven Pontania sawfly species that induce leaf galls on eight willow species growing in subarctic and arctic–alpine habitats in three geographic locations in northern Fennoscandia, and then applied distance‐ and model‐based multivariate analyses and phylogenetic regression methods to evaluate the hierarchical importance of location, phylogeny and different galler niche dimensions on parasitoid host use. We found statistically significant variation in parasitoid communities across geographic locations and willow host species, but the differences were mainly quantitative due to extensive sharing of enemies among gallers within habitat types. By contrast, the divide between habitats defined two qualitatively different network compartments, because many common parasitoids exhibited strong habitat preference. Galler and parasitoid phylogenies did not explain associations, because distantly related arctic–alpine gallers were attacked by a species‐poor enemy community dominated by two parasitoid species that most likely have independently tracked the gallers’ evolutionary shifts into the novel habitat. Our results indicate that barcode‐ and phylogeny‐based analyses of food webs that span forested vs. tundra or grassland environments could improve our understanding of vertical diversification effects in complex plant–herbivore–parasitoid networks.     

Ultrastructural and Immunocytochemical Characterization of Autophagic Vacuoles in Isolated Hepatocytes: Effects of Vinblastine and Asparagine on Vacuole Distributions

The interactions between the autophagic and the endocytic degradation pathways were investigated by means of immunogold labeling of autophagic vacuoles (AVs) in ultrathin frozen sections from isolated rat hepatocytes. AVs were identified by their autophagocytosed contents of the degradation-resistant cytosolic enzyme CuZn-superoxide dismutase (SOD). Another cytosolic enzyme, carbonic anhydrase (CAIII), was rapidly degraded in the lysosomes, making the vacuolar CAIII/SOD ratio useful as a rough indicator of the progress of autophagic-lysosomal degradation. Lysosomes could be recognized by the presence of the lysosomal membrane glycoprotein lgp120, which was absent from hepatocytic endosomes. Endocytic inputs into the AVs were detected by the presence of gold-conjugated bovine serum albumin (BSA-gold), taken up by fluid-phase endocytosis. All vacuoles recognized morphologically as AVs were SOD-positive, as were essentially all of the lysosomes (96%). The majority (72%) of the lysosomes also labeled positively for BSA within 2 h of endocytosis. The data are thus compatible with the notion that all lysosomes can engage in both autophagic and endocytic degradation. Lgp120 appeared to distinguish well between lysosomes and nonlysosomal AVs: the lgp120-negative AVs (nonlysosomes) had a CAIII/SOD ratio identical to that of the cytosol, indicating that no degradation had occurred, In the lgp120-positive AVs (lysosomes), the ratio was only 43% of the cytosolic value, consistent with substantial CAIII degradation. Among the nonlysosomal AVs (about one-third of all AVs), one-half were BSA-positive, suggesting that early AVs (autophagasomes) and intermediary AVs (amphisomes) that had fused with endosomes were equally abundant. These morphological data thus support previous biochemical evidence for a prelysosomal meeting of the autophagic and endocytic pathways. The microtubule inhibitor vinblastine inhibited the autophagic influx to the lysosomes, causing an accumulation of autophagosomes and a reduction in average lysosomal size. Vinblastine also inhibited the endocytic flux, thereby precluding the formation of amphisomes and of BSA-positive lysosomes. High concentrations (20 mM) of asparagine induced swelling of amphisomes and of BSA-positive lysosomes, probably reflecting an acidotropic effect of ammonia generated by asparagine deamination. Asparagine also caused an accumulation of autophagosomes, amphisomes, and BSA-negative lysosomes, presumably as a result of impaired fusion with the swollen BSA-positive lysosomes. The two agents thus appear to perturb the autophagic-endocytic-lysosomal vacuole dynamics by different mechanisms, making them useful in the further study of these complex organelle interactions.     

Molecular modelling,synthesis, and biological evaluations of a 3,5-disubstituted isoxazole fatty acid analogue as a PPARα-selective agonist

The peroxisome proliferator activated receptors (PPARs) are important drug targets in treatment of metabolic and inflammatory disorders. Fibrates, acting as PPARα agonists, have been widely used lipid-lowering agents for decades. However, the currently available PPARα targeting agents show low subtype-specificity and consequently a search for more potent agonists have emerged. In this study, previously isolated oxohexadecenoic acids from the marine algae Chaetoceros karianus were used to design a PPARα-specific analogue. Herein we report the design, synthesis, molecular modelling studies and biological evaluations of the novel 3,5-disubstituted isoxazole analogue 6-(5-heptyl-1,2-oxazol-3-yl)hexanoic acid (1), named ADAM. ADAM shows a clear receptor preference and significant dose-dependent activation of PPARα (EC₅₀ = 47 µM) through its ligand-binding domain (LBD). Moreover, ADAM induces expression of important PPARα target genes, such as CPT1A, in the Huh7 cell line and primary mouse hepatocytes. In addition, ADAM exhibits a moderate ability to regulate PPARγ target genes and drive adipogenesis. Molecular modelling studies indicated that ADAM docks its carboxyl group into opposite ends of the PPARα and -γ LBD. ADAM interacts with the receptor-activating polar network of amino acids (Tyr501, His447 and Ser317) in PPARα, but not in PPARγ LBD. This may explain the lack of PPARγ agonism, and argues for a PPARα-dependent adipogenic function. Such compounds are of interest towards developing new lipid-lowering remedies.     

ATG8 Family Proteins Act as Scaffolds for Assembly of the ULK Complex: SEQUENCE REQUIREMENTS FOR LC3-INTERACTING REGION (LIR) MOTIFS*

Autophagy is a lysosome-dependent degradation system conserved among eukaryotes. The mammalian Atg1 homologues, Unc-51 like kinase (ULK) 1 and 2, are multifunctional proteins with roles in autophagy, neurite outgrowth, and vesicle transport. The mammalian ULK complex involved in autophagy consists of ULK1, ULK2, ATG13, FIP200, and ATG101. We have used pulldown and peptide array overlay assays to study interactions between the ULK complex and six different ATG8 family proteins. Strikingly, in addition to ULK1 and ULK2, ATG13 and FIP200 interacted with human ATG8 proteins, all with strong preference for the GABARAP subfamily. Similarly, yeast and Drosophila Atg1 interacted with their respective Atg8 proteins, demonstrating the evolutionary conservation of the interaction. Use of peptide arrays allowed precise mapping of the functional LIR motifs, and two-dimensional scans of the ULK1 and ATG13 LIR motifs revealed which substitutions that were tolerated. This information, combined with an analysis of known LIR motifs, provides us with a clearer picture of sequence requirements for LIR motifs. In addition to the known requirements of the aromatic and hydrophobic residues of the core motif, we found the interactions to depend strongly on acidic residues surrounding the central core LIR motifs. A preference for either a hydrophobic residue or an acidic residue following the aromatic residue in the LIR motif is also evident. Importantly, the LIR motif is required for starvation-induced association of ULK1 with autophagosomes. Our data suggest that ATG8 proteins act as scaffolds for assembly of the ULK complex at the phagophore.