Casein kinase 1 regulates connexin-43 gap junction assembly

Phosphorylation of members of the connexin family of gap junction proteins has been correlated with gap junction assembly, but the mechanisms involved remain unclear. We have examined the role of casein kinase 1 (CK1) in connexin-43 (Cx43) gap junction assembly. Cellular co-immunoprecipitation experiments and in vitro CK1 phosphorylation reactions indicate that CK1 interacted with and phosphorylated Cx43, initially on serine(s) 325, 328, or 330. (32)P(i)-Metabolically labeled cells treated with CKI-7, a specific CK1 inhibitor, showed a reduction in Cx43 phosphorylation on site(s) that can be phosphorylated by CK1 in vitro. To examine CK1 function, normal rat kidney cells were treated with CKI-7, and Cx43 content was analyzed by Triton X-100 extraction, cell-surface biotinylation, and immunofluorescence. Western blot analysis indicated a slight increase in total Cx43, whereas gap junctional (Triton-insoluble) Cx43 decreased, and non-junctional plasma membrane Cx43 increased (as detected by cell surface biotinylation). Immunofluorescence experiments in the presence of CK1 inhibitor showed increases in Cx43 plasma membrane localization but not necessarily accumulation at cell-cell interfaces. Decreased gap junctional and phosphorylated Cx43 was also detected when cells were treated with IC261, a CK1 inhibitor specific for delta or epsilon isoforms. These data suggest CK1delta could regulate Cx43 gap junction assembly by directly phosphorylating Cx43.     

2-Phenylethylamine catabolism by Escherichia coli K12

Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid. Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation. The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase. Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.     

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.     

Abnormal fertilization is responsible for reduced fecundity following thiram-induced ovulatory delay in the rat

Brief exposure to some pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH and, thus, delay ovulation. Previously, we have shown that a single i.p. injection of 50 mg/kg of thiram, a dithiocarbamate fungicide that decreases norepinephrine synthesis, on proestrus (1300 h) suppresses the LH surge and delays ovulation for 24 h without altering the number of oocytes released. However, when bred, the treated dams had a decreased litter size and increased postimplantation loss. We hypothesized that the reduced litter size in thiram-delayed rats was a consequence of altered oocyte function arising from intrafollicular oocyte aging. To test this hypothesis, we examined delayed oocytes, zygotes, and 2-cell embryos for evidence of fertilization and polyspermy. In addition, we used confocal laser-scanning microscopy to evaluate and characterize cortical granule localization in oocytes and release in zygotes, because the cortical granule response is a major factor in the normal block to polyspermy. Our results demonstrate that a thiram-induced, 24-h delay in ovulation alters the fertilizability of the released oocyte. Although no apparent morphological differences were observed in the unfertilized mature oocytes released following the thiram-induced delay, the changes observed following breeding include a significant decrease in the percentage of fertilized oocytes, a significant increase in polyspermic zygotes (21%), and a 10-fold increase in the number of supernumerary sperm in the perivitelline space. Importantly, all the polyspermic zygotes exhibited an abnormal pattern of cortical granule exudate, suggestive of a relationship between abnormal cortical reaction and the polyspermy in the delayed zygotes. Because polyspermy is associated with polyploidy, abnormal development, and early embryonic death, the observed polyspermy could explain the abnormal development and decreased litter size that we observed previously following thiram-delayed ovulation.     

Avian Incubation Patterns Reflect Temporal Changes in Developing Clutches

Incubation conditions for eggs influence offspring quality and reproductive success. One way in which parents regulate brooding conditions is by balancing the thermal requirements of embryos with time spent away from the nest for self-maintenance. Age related changes in embryo thermal tolerance would thus be expected to shape parental incubation behavior. We use data from unmanipulated Black-capped Chickadee (Poecile atricapillus) nests to examine the temporal dynamics of incubation, testing the prediction that increased heat flux from eggs as embryos age influences female incubation behavior and/or physiology to minimize temperature fluctuations. We found that the rate of heat loss from eggs increased with embryo age. Females responded to increased egg cooling rates by altering incubation rhythms (more frequent, shorter on- and off- bouts), but not brood patch temperature. Consequently, as embryos aged, females were able to increase mean egg temperature and decrease variation in temperature. Our findings highlight the need to view full incubation as more than a static rhythm; rather, it is a temporally dynamic and finely adjustable parental behavior. Furthermore, from a methodological perspective, intra- and inter-specific comparisons of incubation rhythms and average egg temperatures should control for the stage of incubation.     

Postcranial morphology and locomotion of the Eocene raoellid Indohyus (Artiodactyla: Mammalia)

Raoellids are small, raccoon-sized Eocene artiodactyls, closely related to archaic cetaceans (archaeocetes) that have poor representation of postcranial elements in the fossil record. Little is known of the aquatic and terrestrial locomotor affinities of raoellids due to the paucity of their fossil record, leaving a critical gap in our understanding of the earliest portion of the artiodactyl marine invasion. To address this gap, a comparative morphological analysis of the postcranial elements was undertaken based on newly recovered elements of the raoellid Indohyus, archaeocetes and extant artiodactyls. Greater than 200 postcranial elements of Indohyus were described, and some limb element cross-sections were visualised via paleohistological thin sections and CT scans. Results show that during terrestrial locomotion, Indohyus probably had a digitigrade posture and mediolaterally stabilised limbs that functioned mostly in flexion and extension within the parasagittal plane. Quantification of midshaft cross-sectional area for some elements of Indohyus showed an osteosclerotic cortex, a skeletal characteristic associated with aquatic behaviours. Indohyus may represent a critical intermediate in the evolution of the cetacean terrestrial-to-aquatic body plan, as it bears gracile postcranial element proportions similar to a terrestrial artiodactyl but also an incipient form of osteosclerosis compared to pakicetid archaeocetes.     

A landscape approach for identifying potential reestablishment sites for extirpated stream fishes: an example with Arctic grayling (Thymallus arcticus) in Michigan

Hydrobiologia - Habitat degradation combined with climate change increases the threat of extinction for stream fishes. In response to these threats, efforts to reestablish species within formerly...     

A metabolomic comparison of mouse models of the Neuronal Ceroid Lipofuscinoses

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of fatal inherited neurodegenerative diseases in humans distinguished by a common clinical pathology, characterized by the accumulation of storage body material in cells and gross brain atrophy. In this study, metabolic changes in three NCL mouse models were examined looking for pathways correlated with neurodegeneration. Two mouse models; motor neuron degeneration (mnd) mouse and a variant model of late infantile NCL, termed the neuronal ceroid lipofuscinosis (nclf) mouse were investigated experimentally. Both models exhibit a characteristic accumulation of autofluorescent lipopigment in neuronal and non neuronal cells. The NMR profiles derived from extracts of the cortex and cerebellum from mnd and nclf mice were distinguished according to disease/wildtype status. In particular, a perturbation in glutamine and glutamate metabolism, and a decrease in γ-amino butyric acid (GABA) in the cerebellum and cortices of mnd (adolescent mice) and nclf mice relative to wildtype at all ages were detected. Our results were compared to the Cln3 mouse model of NCL. The metabolism of mnd mice resembled older (6?month) Cln3 mice, where the disease is relatively advanced, while the metabolism of nclf mice was more akin to younger (1-2?months) Cln3 mice, where the disease is in its early stages of progression. Overall, our results allowed the identification of metabolic traits common to all NCL subtypes for the three animal models.