A mitochondrial DNA phylogeny of African clawed frogs: phylogeography and implications for polyploid evolution

The African clawed frogs (Silurana and Xenopus), model organisms for scientific inquiry, are unusual in that allopolyploidization has occurred on multiple occasions, giving rise to tetraploid, octoploid, and dodecaploid species. To better understand their evolution, here we estimate a mitochondrial DNA phylogeny from all described and some undescribed species. We examine the timing and location of diversification, and test hypotheses concerning the frequency of polyploid speciation and taxonomy. Using a relaxed molecular clock, we estimate that extant clawed frog lineages originated well after the breakup of Gondwana, about 63.7 million years ago, with a 95% confidence interval from 50.4 to 81.3 million years ago. Silurana and two major lineages of Xenopus have overlapping distributions in sub-Saharan Africa, and dispersal-vicariance analysis suggests that clawed frogs originated in central and/or eastern equatorial Africa. Most or all extant species originated before the Pleistocene; recent rainforest refugia probably acted as "lifeboats" that preserved existing species, rather than "species pumps" where many new successful lineages originated. We estimate that polyploidization occurred at least six times in clawed frogs.     

Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43

We have previously shown in cultured rat microvascular endothelial cells (RMEC) that lipopolysaccharide (LPS) stimulates a protein tyrosine kinase (PTK)-dependent reduction in cellular coupling. We hypothesized that connexin 43 (Cx43) becomes phosphorylated following exposure to LPS. Cx43 was immunoprecipitated from control and LPS-treated RMEC monolayers. Tyrosine phosphorylation of Cx43, detected by immunoblot, was found only in the LPS treatment. To verify these results, Cx43 was radiolabeled with [(32)P]-orthophosphate. Radiolabeled Cx43 exhibited a slight increase in phosphorylation in response to LPS; phosphoamino acid analysis displayed equivalent amounts of phosphoserine in control and LPS treatments, but detected phosphotyrosine only in the LPS treatment. The PTK inhibitors PP-2 (10 nM) and geldanamycin (200 nM) were found to block the response to LPS in terms of Cx43 tyrosine phosphorylation and cellular coupling. The phosphatase inhibitor BpV (1 microM) accentuated the effect of LPS, while the putative phosphatase activator C(6)-ceramide prevented it. When measuring cell communication, phosphatase inhibition also blocked the reversal of the LPS response following LPS washout. We conclude that Cx43 is tyrosine phosphorylated following exposure to LPS and suggest that the LPS-induced increase in intercellular resistance may be mediated by tyrosine phosphorylation of this connexin. Altering tyrosine kinase and phosphatase activities can modulate the LPS-induced tyrosine phosphorylation of Cx43 and reductions in cellular coupling.     

Neuronal differentiation and growth control of neuro-2a cells after retroviral gene delivery of connexin43

Given the roles proposed for gap junctional intercellular communication in neuronal differentiation and growth control, we examined the effects of connexin43 (Cx43) expression in a neuroblastoma cell line. A vesicular stomatitis virus G protein (VSVG)-pseudotyped retrovector was engineered to co-express the green fluorescent protein (GFP) and Cx43 in the communication-deficient neuro-2a (N2a) cell line. The 293 GPG packaging cell line was used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or chimeric Cx43.GFP fusion protein. The titer of viral supernatant, as measured by flow cytometry for GFP fluorescence, was approximately 2.0 x 10(7) colony form units (CFU)/ml and was free of replication-competent retroviruses. After a 7-day treatment with retinoic acid (20 microm), N2a transformants (N2a-Cx43 and N2a-Cx43.GFP) maintained the expression of Cx43 and Cx43.GFP. Expression of both constructs resulted in functional coupling, as evidenced by electrophysiological and dye-injection analysis. Suppression of cell growth correlated with expression of both Cx43 or Cx43.GFP and retinoic acid treatment. Based on morphology and immunocytochemistry for neurofilament, no difference was observed in the differentiation of N2a cells compared with cells expressing Cx43 constructs. In conclusion, constitutive expression of Cx43 in N2a cells does not alter retinoic acid-induced neuronal differentiation but does enhance growth inhibition.     

Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene

The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2^+/- mice appear to develop in a similar manner to wild type mice (Her2^-/-), it has proven difficult to generate homozygous Her2^+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2^+/+ mice, similar to Pds5b^-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.     

Genetics,Morphology, Advertisement Calls,and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus,Pipidae) from West and Central Africa

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).     

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography,Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.     

Grazers and vitamins shape chain formation in a bloom-forming dinoflagellate, Cochlodinium polykrikoides

Predators influence the phenotype of prey through both natural selection and induction. We investigated the effects of grazers and nutrients on chain formation in a dinoflagellate, Cochlodinium polykrikoides, which forms dense blooms and has deleterious effects on marine ecosystems around the world. Field populations of C. polykrikoides formed longer chains than laboratory cultures without grazers. In the field, chain length of C. polykrikoides was positively correlated with the abundance of the copepod Acartia tonsa. Chain length of C. polykrikoides increased when exposed to live females of A. tonsa or its fresh (<24 h post-isolation) exudates for 48 h. These results suggest that dissolved chemical cues released by A. tonsa induce chain formation in C. polykrikoides. Ingestion rate of A. tonsa on four-cell chains of C. polykrikoides was lower than on single cells, suggesting that chain formation may be an effective anti-grazing defense. Finally, nutrient amendment experiments demonstrated that vitamins (B₁, B₇, and B₁₂) increased the chain length of C. polykrikoides both singly and collectively, while trace metals and inorganic nutrients did not, showing that vitamins may also influence chain formation in this species.     

Reproduction of in vivo motion using a parallel robot

Although alterations in knee joint loading resulting from injury have been shown to influence the development of osteoarthritis, actual in vivo loading conditions of the joint remain unknown. A method for determining in vivo ligament loads by reproducing joint specific in vivo kinematics using a robotic testing apparatus is described. The in vivo kinematics of the ovine stifle joint during walking were measured with 3D optical motion analysis using markers rigidly affixed to the tibia and femur. An additional independent single degree of freedom measuring device was also used to record a measure of motion. Following sacrifice, the joint was mounted in a robotic/universal force sensor test apparatus and referenced using a coordinate measuring machine. A parallel robot configuration was chosen over the conventional serial manipulator because of its greater accuracy and stiffness. Median normal gait kinematics were applied to the joint and the resulting accuracy compared. The mean error in reproduction as determined by the motion analysis system varied between 0.06 mm and 0.67 mm and 0.07 deg and 0.74 deg for the two individual tests. The mean error measured by the independent device was found to be 0.07 mm and 0.83 mm for the two experiments, respectively. This study demonstrates the ability of this system to reproduce in vivo kinematics of the ovine stifle joint in vitro. The importance of system stiffness is discussed to ensure accurate reproduction of joint motion.     

A protein kinase Cepsilon-anti-apoptotic kinase signaling complex protects human vascular endothelial cells against apoptosis through induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.