Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca2+-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKKbeta using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKKbeta was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKKbeta or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca2+ as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca2+/CaMKKbeta dependent and one AMP/LKB1 dependent.     

Indigenous Knowledge of Rock Kangaroo Ecology in Western Arnhem Land, Australia

Indigenous peoples of western Arnhem Land, central northern Australia, have detailed knowledge of the rock kangaroos of the region, species that are little known to science. Information about the ecology of the species is required for their conservation and management. Ethnoecological studies can assist senior indigenous people with transfer of knowledge and can give respect and meaningful employment to those involved. We used semidirected interviews in the regional vernacular, Bininj Kunwok, to record indigenous knowledge of the ecology of the four rock kangaroo species (Petrogale brachyotis, P. concinna, Macropus bernardus and M. robustus). Discussions focussed on habitat preferences, diet, activity patterns, reproduction, predation, and hunting practices. The ethnoecological knowledge of the rock kangaroo species was extensive, and both complemented and extended that reported in the scientific literature. In contrast to scientific understanding of taxonomy and ecology, consultants recognized the rock kangaroos as a natural group. They also described subtle differences in the species’ comparative ecology. The methodology used proved highly successful and we recommend recording indigenous knowledge of the ecology of fauna species in the local vernacular wherever possible. This study is one of the most comprehensive ethnozoological studies of a group of species undertaken in Australia.

Simulating complex tumor dynamics from avascular to vascular growth using a general level-set method

A comprehensive continuum model of solid tumor evolution and development is investigated in detail numerically, both under the assumption of spherical symmetry and for arbitrary two-dimensional growth. The level set approach is used to obtain solutions for a recently developed multi-cell transport model formulated as a moving boundary problem for the evolution of the tumor. The model represents both the avascular and the vascular phase of growth, and is able to simulate when the transition occurs; progressive formation of a necrotic core and a rim structure in the tumor during the avascular phase are also captured. In terms of transport processes, the interaction of the tumor with the surrounding tissue is realistically incorporated. The two-dimensional simulation results are presented for different initial configurations. The computational framework, based on a Cartesian mesh/narrow band level-set method, can be applied to similar models that require the solution of coupled advection-diffusion equations with a moving boundary inside a fixed domain. The solution algorithm is designed so that extension to three-dimensional simulations is straightforward.     

Trace fossils as indicators of environmental change in Holocene sediments of the Archipelago Sea, northern Baltic Sea

Biogenic structures in Holocene sediments from the Archipelago Sea, northern Baltic Sea, were characterized through the analysis of X-ray images of four cores collected from water depths between 32 and 66 m. In the area, initial colonization of endobenthic invertebrates occurred at 7800 ± 80 calendar years BP. The trace assemblage at this level is low-diversity, small-diameter, shallowly tiered, and Palaeophycus-dominated; rare Arenicolites are also observed. Early colonization coincides with increasing marine influence in the post-glacial lacustrine setting just before the dramatic onset of brackish-water conditions established after 7600 BP. The post-incursion brackish-water assemblage possesses a higher diversity of traces (Planolites, Arenicolites, Lockeia, Teichichnus), which is taken to reflect the enhanced salinity and trophic state of the basin. The shift from Palaeophycus-mottling to a Planolites-dominated fabric represents changed behavioural patterns in the endobenthic community due to changed substrate properties. The ethology of the succeeding trace assemblage also represents a switch from domicile-based activities, such as predation, scavenging and interface-dominated deposit feeding to shallow-tier deposit feeding. Finally, traces are excluded from thinly laminated intervals, demonstrating that seafloor oxygen deficiency commonly reached levels that were detrimental to colonization of the sediment substrate.     

Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subsp. avium infections in a tule elk (Cervus elaphus nannodes) herd

Between 2 August and 22 September 2000, 37 hunter-killed tule elk (Cervus elaphus nannodes) were evaluated at the Grizzly Island Wildlife Area, California, USA, for evidence of paratuberculosis. Elk were examined post-mortem, and tissue and fecal samples were submitted for radiometric mycobacterial culture. Acid-fast isolates were identified by a multiplex polymerase chain reaction (PCR) that discriminates among members of the Mycobacterium avium complex (MAC). Histopathologic evaluations were completed, and animals were tested for antibodies using a Johne's enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion. In addition, 104 fecal samples from tule elk remaining in the herd were collected from the ground and submitted for radiometric mycobacterial culture. No gross lesions were detected in any of the hunter-killed animals. Mycobacterium avium subsp. paratuberculosis (MAP) was cultured once from ileocecal tissue of one adult elk and was determined to be a strain (A18) found commonly in infected cattle. One or more isolates of Mycobacterium avium subsp. avium (MAA) were isolated from tissues of five additional adult elk. Gastrointestinal tract and lymph node tissues from 17 of the 37 elk (46%) examined had histopathologic lesions commonly seen with mycobacterial infection; however, acid-fast bacteria were not observed. All MAC infections were detected from adult elk (P = 0.023). In adult elk, a statistically significant association was found between MAA infection and ELISA sample-to-positive ratio (S/P) > or = 0.25 (P=0.021); four of five MAA culture-positive elk tested positive by ELISA. Sensitivity and specificity of ELISA S/P > or = 0.25 for detection of MAA in adult elk were 50% and 93%, respectively. No significant associations were found between MAC infection and sex or histopathologic lesions. Bacteriologic culture confirmed infection with MAP and MAA in this asymptomatic tule elk herd. The Johne's ELISA was useful in signaling mycobacterial infection on a population basis but could not discriminate between MAA and MAP antibodies. The multiplex PCR was useful in discriminating among the closely related species belonging to MAC.     

Spermatogenesis in the turkey (Meleagris gallopavo): quantitative approach in immature and adult males subjected to various photoperiods

The objectives of this study were to identify and quantitate the germ cell populations of the testes in sexually mature male turkeys (Trial 1), determine the duration of meiosis based on BrdU labeling and stereological analyses (Trial 2), and examine the impact of various photoperiods on germinal and somatic cell populations in immature and adult males (Trial 3). In Trial 1, both testes within a male had similar stereological components (P>0.05) for all parameters analyzed. In Trial 2, the duration of Type-1 spermatocytes and round spermatids in turkeys lasted 4.5+/-0.5 and 2.0+/-0.5 days, respectively. In Trial 3, the short photoperiod (7L:17D) delayed testicular growth (in the stereological parameters analyzed). In contrast, the effect of a moderately short photoperiod (10.5L:13.5D) was comparable to the effect of a long (14L:10D) or increasing photoperiod (7L:17D to 14L:10D) on the stereological parameters examined. With the exception of the short photoperiod, all other photoperiods used in this study induced comparable early testicular maturation, with maximum testis weight at 29-35 weeks of age. As the males got older, there was a progressive, linear decline in testis weight through 60 weeks, at which time there were no significant differences among photoperiods. In conclusion, the duration of meiosis in the turkey was similar to that observed in the fowl and guinea-fowl. The existence of a threshold of photosensitivity to gonad stimulation in male turkeys is suggested to be between 7.0 and 10.5 h of light.     

Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a

The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.     

The Wnt signaling pathway has tumor suppressor properties in retinoblastoma

Retinoblastoma is a pediatric retinal tumor caused by mutational inactivation of the tumor suppressor pRb. Additional genetic changes, as yet unidentified, are believed to be required for tumor initiation. Mutations in the Wnt signaling pathway have been implicated in the pathogenesis of many cancers. Multiple Wnt pathway genes are expressed in the retina and the pRb and Wnt pathways interact biochemically, raising the possibility that alterations in the Wnt pathway contribute to retinoblastoma. Our studies showed that Wnt signaling activation significantly decreased the viability of retinoblastoma cell lines by inducing cell cycle arrest, which was associated with upregulated p53. Furthermore, immunolocalization of the Wnt signaling mediator beta-catenin in human and mouse retinoblastoma tissue indicated that canonical Wnt signaling is suppressed in tumors in vivo. These studies are consistent with the Wnt pathway acting as a tumor suppressor in retinoblastoma and suggest that loss of Wnt signaling is tumorigenic in the retina.