Neuronal ceroid lipofuscinosis related ER membrane protein CLN8 regulates PP2A activity and ceramide levels

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN8 deficiency causes a subtype of NCL, referred to as CLN8 disease. CLN8 is an ER resident protein with unknown function; however, a role in ceramide metabolism has been suggested. In this report, we identified PP2A and its biological inhibitor I2PP2A as interacting proteins of CLN8. PP2A is one of the major serine/threonine phosphatases in cells and governs a wide range of signaling pathways by dephosphorylating critical signaling molecules. We showed that the phosphorylation levels of several substrates of PP2A, namely Akt, S6 kinase, and GSK3β, were decreased in CLN8 disease patient fibroblasts. This reduction can be reversed by inhibiting PP2A phosphatase activity with cantharidin , suggesting a higher PP2A activity in CLN8-deficient cells. Since ceramides are known to bind and influence the activity of PP2A and I2PP2A, we further examined whether ceramide levels in the CLN8-deficient cells were changed. Interestingly, the ceramide levels were reduced by 60% in CLN8 disease patient cells compared to controls. Furthermore, we observed that the conversion of ER-localized NBD-C6-ceramide to glucosylceramide and sphingomyelin in the Golgi apparatus was not affected in CLN8-deficient cells, indicating transport of ceramides from ER to the Golgi apparatus was normal. A model of how CLN8 along with ceramides affects I2PP2A and PP2A binding and activities is proposed.     

A study on the cytoplasmic granules of the pericardial gland cells of some bivalve molluscs

The pericardial glands of three bivalve molluscs are composed of convoluted epithelium that appears as pouches on the auricles of Mytilus and as tubules in the connective tissue at the anterior-lateral sides of the pericardial cavity of Mercenaria and Anodonta. The pericardial gland cells are attached to each other by many randomly placed desmosome-like cell junctions and gap junctions. Belt-desmosomes that are characteristic of epithelial cells were not observed. The basal membrane of these cells is invaginated producing complex interdigitating cytoplasmic processes and filtration slits. The pericardial gland cells stain for the presence of iron with Prussian blue stain. Electron-dense and electron-lucent granules of various diameters are present in the cytoplasm. Many electron-dense granules contain ferritin-like particles in which the presence of iron has been demonstrated by microanalysis. It is suggested that these particles are the iron storage protein ferritin since they contain iron, and are water soluble, heat stable, and morphologically similar to mammalian ferritin. Ferritin particles are probably both synthesized and broken down by the pericardial gland cells; thus the pericardial gland cells may be involved in iron homeostasis in these molluscs.     

Is mass loss in Brünnich's guillemots Uria lomvia an adaptation for improved flight performance or improved dive performance?

Breeding Brünnich's guillemots Uria lomvia show stepwise mass loss at the time of hatch. This mass loss has usually been explained as an adaptation to reduce the cost of flight during the chick‐rearing period because flight time increases during that period. It is possible, however, that mass loss also increases dive performance during the chick‐rearing period because time spent diving also increases during that period. Reduced mass could reduce basal metabolic rate or costs associated with buoyancy and therefore increase aerobic dive limit. To examine the role of mass loss in dive behavior, we attached time‐depth‐temperature recorders for 24–48 h to chick‐rearing and incubating Brünnich's guillemots at Coats Island, Nunavut (2005: n=45, 2006: n=40), and recorded body mass before and after each deployment. There was no relationship between mass and dive duration during either incubation or chick‐rearing. Seventeen of the birds we sampled during incubation were resampled during chick‐rearing. For this group, dive duration increased with mass loss between incubation and chick‐rearing (r²=0.67–0.75). Mass loss occurred through reductions in metabolically‐active tissues (liver, bladder) and buoyant tissues (lipids) although muscle and gut mass did not change. Despite the large change in lipids, buoyancy only changed by 0.1%, and mass loss therefore did not have much effect on costs associated with buoyancy. Nonetheless, surface pause duration for a given dive depth decreased during chick‐rearing, supporting the idea that reduced mass led to increased aerobic dive limit through reduced metabolic rate and inertial costs; oxygen stores did not increase. We also attached neutrally (n=9) and negatively (n=11) buoyant handicaps to the legs of adults to assess the effect of artificial mass increases on time budgets. Artificially increasing mass decreased total time spent diving but did not change time spent flying. There was no change in shift length between incubation and chick‐rearing, and therefore no support for the idea that mass loss reflected a change in fasting endurance requirements. An energetic model suggested that the observed mass reduction reduced dive costs by 5–8% and flight costs by 3%. We concluded that mass loss may be as important for increasing dive performance as increasing flight performance.     

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells II. Test validation and interpretation

The L5178Y Mouse Lymphoma TK assay was studied extensively to determine if this mammalian cell assay for gene mutations at the thymidine kinase (TK) locus could provide valid, interpretable determinations of mutagenic potential, and whether this information is of value in the safety evaluation of chemicals. We first determined that test-derived TFT^R mutants were phenotypically stable, possessing little or no thymidine kinase activity as measured by labeled thymidine uptake, but demonstrating 100% cross resistance to bromodeoxyuridine. Common solvent vehicles such as acetone, dimethylsulfoxide and ethanol were shown to produce little cytotoxicity and no mutagenic activity when present at 1% levels. Out of a total of 10 noncarcinogens tested, all were negative when results were analyzed by a 2-sample log_et test on control and treated mutant count means. Of the 13 putative animal carcinogens tested, 10 were positive, 2 were negative (auramine O and sodium phenobarbital), and 1 showed sporadic activity (hydrazine sulfate) in the TK assay on the basis of test-derived t statistics. 2 compounds, 1,2-epoxybutane and ICR 191, which have been described as Ames positive non-carcinogens, were also positive in the TK assay. Although this sampling of a total of 29 compounds is insufficient for precise estimations of expected false-positive or false-negative frequencies, these data indicate the TK assay can be expected to detect a majority of carcinogens as mutagens including some missed by more established point-mutation assays.     

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development

Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development.     

Differences in the infectivity of Babesia incubated in plasma and serum and the role of glucose in prolonging viability

Babesia rodhaini was less infective to mice after incubation in rat serum than in rat plasma. This was explained by lower levels of glucose in serum than in plasma. Both serum and plasma were found to become metabolically depleted of glucose following prolonged contact with clotted and unclotted blood cells, respectively. When glucose concentrations in depleted samples were restored to those in freshly separated samples, infectivities of parasites were similar. This was shown for both B. rodhaini and B. argentina. Products of blood cell metabolism, produced when separation of plasma or serum was delayed for 24 h, were not shown to have any detrimental effects on the parasites. Average glucose values for plasma from rats and cattle were 153 mg/100 ml and 63 mg/100 ml, respectively, whereas serum and plasma remaining in contact with blood cells contained as little as 2 mg glucose/100 ml. Lactate values were correspondingly low in plasma and high in serum. Fibrinogen and platelets, factors involved in clotting, did not affect infectivity of B. rodhaini or B. argentina. The relevance of these findings to living babesial vaccines in which plasma- and serum-based diluents may be used is discussed.     

Heparin-binding epidermal growth factor-like growth factor inhibits cytokine-induced NF-kappa B activation and nitric oxide production via activation of the phosphatidylinositol 3-kinase pathway

NO produced by inducible NO synthase (iNOS) has been implicated in various pathophysiological processes including inflammation. Therefore, inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-inflammatory agents. We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) decreases proinflammatory cytokine IL-8 and NO production in cytokine-stimulated intestinal epithelial cells by interfering with the NF-kappaB signaling pathway. However, the upstream signaling mechanisms involved in these responses have not yet been defined. In this report, we show that in intestinal epithelial cells, HB-EGF triggered PI3K-dependent phosphorylation of Akt. Inhibition of PI3K reversed the ability of HB-EGF to block NF-kappaB activation, expression of iNOS, and NO production. Small interfering RNA of PI3K also reversed the inhibitory effect of HB-EGF on iNOS expression. Alternatively, transient expression of constitutively active PI3K decreased NO production by approximately 2-fold more than treatment with HB-EGF alone. This PI3K effect was HB-EGF dependent. Thus, activation of PI3K is essential but not sufficient for decreased NO synthesis. PI3K and HB-EGF act synergistically to decrease NO synthesis. Neither overexpression or inhibition of MEK, Ras, or Akt affected HB-EGF-mediated inhibition of NF-kappaB activation. These data demonstrate that HB-EGF decreases proinflammatory cytokine-stimulated NF-kappaB activation and NO production via activation of the PI3K signaling pathway. These results also suggest that inhibition of NF-kappaB and activation of the PI3K-dependent signaling cascade by HB-EGF may represent key signals responsible for the anti-inflammatory effects of HB-EGF.     

Growth,nutrient acquisition and ectomycorrhizae of <Emphasis Type="Italic">Eucalyptus regnans</Emphasis> F. Muell. seedlings in fertilized or diluted air-dried and undried forest soil

Seedlings of Eucalyptus regnans (mountain ash) grow poorly in undried forest soil, where they develop purple coloration in the foliage, but their growth is markedly improved when forest soil has been air dried. Whether this growth promotion is purely due to improved nutrient status of the soil, as a result of air drying, was investigated. In several pot experiments, E. regnans seedlings were grown (i) in air-dried and undried forest soil with addition of different levels of complete fertiliser, (ii) in air-dried or undried soil diluted to different extents with sand, or (iii) in undried soil mixed with different amounts of air-dried soil. Seedling dry weight, P content and incidence of ectomycorrhizal root tips were determined.In all experiments, the dry weights of seedlings were 3–6 times greater in 100% air-dried soil than in 100% undried soil. Fertiliser application resulted in a significant increase in dry weight of seedlings in both air-dried and undried soil, but the dry weights in air-dried soil were always significantly greater than those in undried soil at the same level of fertiliser application. Even at the highest level of fertiliser application, the growth difference between seedlings in air-dried and undried soil remained. When air-dried soil was diluted with sand, there was a significant reduction in seedling dry weight only when soil was diluted to 20% or less (air-dried soil:total mix). Conversly, when air-dried soil was mixed with undried soil, there was a proportional decrease in seedling dry weight with increasing amounts of undried soil. In all experiments, the dominant ectomycorrhizal morphotypes in 100% air-dried soil were different from those in undried soil. Fertilisation and dilution of air-dried and undried soil did not result in a reduction in the overall incidence of ectomycorrhizal root tips, although the frequency of occurrence of different ectomycorrhizal morphotypes was affected.It is concluded that the growth difference between seedlings in air-dried and undried forest soils is not due solely to differences in the direct availability of nutrients in the soils, and different ectomycorrhizae may indirectly affect nutrient availability to the plant.     

Terpenoid aldehyde formation and lysigenous gland storage sites in cotton: variant with mature glands but suppressed levels of terpenoid aldehydes

A new cotton variant with reduced levels of terpenoid aldehydes (sesquiterpenoids and sesterterpenoids (heliocides)) was isolated from the progeny of hemizygous cotton (Gossypium hirsutum cv. Coker 312) transformed with antisense (+)-delta-cadinene synthase cDNA. Southern analysis of leaf DNA digested with HindIII, Pst or KpnI restriction endonucleases did not detect any antisense cdn1-C1 DNA in the genome of the variant. The gossypol content in the seed of the variant was markedly lower than in the seed of T1 antisense plants. Eighty-nine percent of the variant seed had a 71.1% reduction in gossypol and the foliage of the variant plants showed a 70% reduction in gossypol and a 31% reduction in heliocides. Compared to non-transformed plants there was no reduction in the number of lysigenous glands in the seed of the variant. The cotton variant shows uncoupling of terpenoid aldehyde synthesis and gland formation. The cotton variant may have resulted from somaclonal variation occurring in the callus tissue during the transformation-regeneration process.     

Bergmann's rule in nonavian reptiles: turtles follow it,lizards and snakes reverse it

Abstract Bergmann's rule is currently defined as a within-species tendency for increasing body size with increasing latitude or decreasing environmental temperature. This well-known ecogeographic pattern has been considered a general trend for all animals, yet support for Bergmann's rule has only been demonstrated for mammals and birds. Here we evaluate Bergmann's rule in two groups of reptiles: chelonians (turtles) and squamates (lizards and snakes). We perform both nonphylogenetic and phylogenetic analyses and show that chelonians follow Bergmann's rule (19 of 23 species increase in size with latitude; 14 of 15 species decrease in size with temperature), whereas squamates follow the converse to Bergmann's rule (61 of 83 species decrease in size with latitude; 40 of 56 species increase in size with temperature). Size patterns of chelonians are significant using both nonphylogenetic and phylogenetic methods, whereas only the nonphylogenetic analyses are significant for squamates. These trends are consistent among major groups of chelonians and squamates for which data are available. This is the first study to document the converse to Bergmann's rule in any major animal group as well as the first to show Bergmann's rule in a major group of ectotherms. The traditional explanation for Bergmann's rule is that larger endothermic individuals conserve heat better in cooler areas. However, our finding that at least one ectothermic group also follows Bergmann's rule suggests that additional factors may be important. Several alternative processes, such as selection for rapid heat gain in cooler areas, may be responsible for the converse to Bergmann's rule in squamates.