Assembly of the eastern North American herpetofauna: new evidence from lizards and frogs

Darwin first recognized the importance of episodic intercontinental dispersal in the establishment of worldwide biotic diversity. Faunal exchange across the Bering Land Bridge is a major example of such dispersal. Here, we demonstrate with mitochondrial DNA evidence that three independent dispersal events from Asia to North America are the source for almost all lizard taxa found in continental eastern North America. Two other dispersal events across Beringia account for observed diversity among North American ranid frogs, one of the most species-rich groups of frogs in eastern North America. The contribution of faunal elements from Asia via dispersal across Beringia is a dominant theme in the historical assembly of the eastern North American herpetofauna.     

Activity patterns and spatial use of facility by a group of captive female manatees (Trichechus manatus latirostris)

As a means to compare and contrast to free‐ranging Florida manatees, we examined the behavior and movement patterns of nine adult‐female captive manatees at Homosassa Springs Wildlife State Park (HSWSP). HSWSP has a unique manatee exhibit that consists of a natural river and a man‐made pool. Because the manatees are exposed to a natural environment including natural food resources, the study of various aspects of behavioral and physiological parameters of the captive manatees is especially valuable with this group. We determined activity pattern and spatial use of the facility by manatees over three allotted periods of a day (noon, mid‐afternoon, and late‐afternoon) and three seasons (winter, spring, and summer). Behavioral strategies of the manatees were: 1) to remain “inactive,” probably to conserve energy, during the day while the park provisioned food; 2) to alter locations during the course of the afternoon, apparently with the direction of the sun, possibly to assist with thermoregulation; and 3) to change location seasonally where and when food resources became abundant, showing a strong interest in natural vegetation as it became available. Spatial use and activity pattern of the HSWSP captive manatees apparently were influenced by energy constraints and nutrient intake by provisioned food availability over the day and natural vegetation over the study period. The behavior of the female group of manatees may have been affected by the single‐sex living arrangement. In addition, these manatees are subject to a largely invariable temperature (ca. 23°C) that may have created thermal stress and immunological suppression over time. This study demonstrated that for these captive manatees the activity patterns and spatial use were comparable to that of free‐ranging manatees in relation to the availability of food resources. Zoo Biol 0:1–17, 2006. © 2006 Wiley‐Liss, Inc.     

C-terminal truncation impairs glycosylation of transmembrane collagen XVII and leads to intracellular accumulation

Collagen XVII, a type II transmembrane protein in hemidesmosomes, is involved in the anchorage of stratified epithelia to the underlying mesenchyme. Its functions are regulated by ectodomain shedding, and its genetic defects lead to epidermal detachment in junctional epidermolysis bullosa (JEB), a heritable skin fragility syndrome, but the molecular disease mechanisms remain elusive. Here we used a spontaneously occurring homozygous COL17A1 deletion mutant in JEB to discern glycosylation of collagen XVII. The mutation truncated the distal ectodomain and positioned the only N-glycosylation site 34 amino acids from the newly formed C terminus, which impaired efficient N-glycosylation. Immunofluorescence staining of authentic JEB keratinocytes and of COS-7 cells transfected with the mutant indicated intracellular accumulation of collagen XVII precursor molecules. Cell surface biotinylation and quantification of ectodomain shedding demonstrated that only about 15% of the truncated collagen XVII reached the cell surface. The cell surface-associated molecules were N-glycosylated in a normal manner, in contrast to the molecules retained within the cells, indicating that N-glycosylation of the ectodomain is required for targeting of collagen XVII to the plasma membrane and that reduced accessibility of the N-glycosylation site negatively regulates this process. Functional consequences of the strong reduction of collagen XVII on the cell surface included scattered deposition of cell adhesion molecule laminin 5 into the extracellular environment and, as a consequence of faulty collagen XVII-laminin ligand interactions, aberrant motility of the mutant cells.     

Interactions of granisetron with an agonist-free 5-HT3A receptor model

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.     

Paternal inheritance in parthenogenetic forms of the planarian Schmidtea polychroa

Parthenogenesis usually includes clonal inheritance, which is thought to increase the risk of the clonal populations' extinction. Yet many parthenogenetic organisms appear to have survived for extended periods. A possible explanation is that parthenogens occasionally reproduce through sex-like processes. Although there is indirect evidence for occasional sex, the underlying mechanisms are currently unknown. In the present study, we examined sex-like processes in the planarian flatworm Schmidtea (Dugesia) polychroa. Parthenogenetic forms of this species are simultaneous hermaphrodites that require sperm to trigger embryogenesis, whereas paternal genetic material is usually excluded from the oocyte (sperm-dependent parthenogenesis). Based on a comparison of parents and offspring, using highly polymorphic microsatellites, we demonstrate the incorporation of paternal alleles in about 5% of the offspring. We detected two distinct processes: chromosome addition and chromosome displacement. Such rare sexual processes may explain the long-term persistence of the many purely parthenogenetic populations of S. polychroa in northern Europe.     

High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes

AMPA-type glutamate receptors (AMPARs) are responsible for a variety of processes in the mammalian brain including fast excitatory neurotransmission, postsynaptic plasticity, or synapse development. Here, with comprehensive and quantitative proteomic analyses, we demonstrate that native AMPARs are macromolecular complexes with a large molecular diversity. This diversity results from coassembly of the known AMPAR subunits, pore-forming GluA and three types of auxiliary proteins, with 21 additional constituents, mostly secreted proteins or transmembrane proteins of different classes. Their integration at distinct abundance and stability establishes the heteromultimeric architecture of native AMPAR complexes: a defined core with a variable periphery resulting in an apparent molecular mass between 0.6 and 1 MDa. The additional constituents change the gating properties of AMPARs and provide links to the protein dynamics fundamental for the complex role of AMPARs in formation and operation of glutamatergic synapses.     

Computational fluid dynamics models of conifer bordered pits show how pit structure affects flow

? The flow of xylem sap through conifer bordered pits, particularly through the pores in the pit membrane, is not well understood, but is critical for an understanding of water transport through trees. ? Models solving the Navier-Stokes equation governing fluid flow were based on the geometry of bordered pits in black spruce (Picea mariana) and scanning electron microscopy images showing details of the pores in the margo of the pit membrane. ? Solutions showed that the pit canals contributed a relatively small fraction of resistance to flow, whereas the torus and margo pores formed a large fraction, which depended on the structure of the individual pit. The flow through individual pores in the margo was strongly dependent on pore area, but also on the radial location of the pore with respect to the edge of the torus. ? Model results suggest that only a few per cent of the pores in the margo account for nearly half of the flow and these pores tend to be located in the inner region of the margo where their contribution will be maximized. A high density of strands in outer portions of the margo (hence narrower pores) may be more significant for mechanical support of the torus.     

Thermal acclimation is not necessary to maintain a wide thermal breadth of aerobic scope in the common killifish (Fundulus heteroclitus)

Loss of aerobic scope at high and low temperatures is a physiological mechanism proposed to limit the thermal performance and tolerance of organisms, a theory known as oxygen- and capacity-limited thermal tolerance (OCLTT). Eurythermal organisms maintain aerobic scope over wide ranges of temperatures, but it is unknown whether acclimation is necessary to maintain this breadth. The objective of this study was to examine changes in aerobic scope in Fundulus heteroclitus, a eurythermal fish, after acclimation and acute exposure to temperatures from 5° to 33°C. The range of temperatures over which aerobic scope was nonzero was similar in acclimated and acutely exposed fish, suggesting that acclimation has modest effects on the thermal breadth of aerobic scope. However, in acclimated fish, there was a clear optimum temperature range for aerobic scope between 25° and 30°C, whereas aerobic scope was relatively constant across the entire temperature range with acute temperature exposure. Therefore, the primary effect of acclimation was to increase aerobic scope between 25° and 30°C, which paradoxically resulted in a narrower temperature range of optimal performance in acclimated fish compared to acutely exposed fish. There was only weak evidence for correlations between the thermal optimum of aerobic scope and the thermal optimum of measures of performance (specific growth rate and gonadosomatic index), and indicators of anaerobic metabolism (lactate accumulation and lactate dehydrogenase activity) only increased at high temperatures. Together these data fit many, but not all, of the predictions made by OCLTT.     

Expression of Eukaryotic Initiation Factor 5A and Hypusine Forming Enzymes in Glioblastoma Patient Samples: Implications for New Targeted Therapies

Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA) could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity.