UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

Intramolecular interaction between the DEP domain of RGS7 and the Gbeta5 subunit

The R7 family of RGS proteins (RGS6, -7, -9, -11) is characterized by the presence of three domains: DEP, GGL, and RGS. The RGS domain interacts with Galpha subunits and exhibits GAP activity. The GGL domain permanently associates with Gbeta5. The DEP domain interacts with the membrane anchoring protein, R7BP. Here we provide evidence for a novel interaction within this complex: between the DEP domain and Gbeta5. GST fusion of the RGS7 DEP domain (GST-R7DEP) binds to both native and recombinant Gbeta5-RGS7, recombinant Gbetagamma complexes, and monomeric Gbeta5 and Gbeta1 subunits. Co-immunoprecipitation and FRET assays supported the GST pull-down experiments. GST-R7DEP reduced FRET between CFP-Gbeta5 and YFP-RGS7, indicating that the DEP-Gbeta5 interaction is dynamic. In transfected cells, R7BP had no effect on the Gbeta5/RGS7 pull down by GST-R7DEP. The DEP domain of RGS9 did not bind to Gbeta5. Substitution of RGS7 Glu-73 and Asp-74 for the corresponding Ser and Gly residues (ED/SG mutation) of RGS9 diminished the DEP-Gbeta5 interaction. In the absence of R7BP both the wild-type RGS7 and the ED/SG mutant attenuated muscarinic M3 receptor-mediated Ca2+ mobilization. In the presence of R7BP, wild-type RGS7 lost this inhibitory activity, whereas the ED/SG mutant remained active. Taken together, our results are consistent with the following model. The Gbeta5-RGS7 molecule can exist in two conformations: "closed" and "open", when the DEP domain and Gbeta5 subunit either do or do not interact. The closed conformation appears to be less active with respect to its effect on Gq-mediated signaling than the open conformation.     

Bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences

We sequenced and analyzed the full-length genomes of four coronaviruses (CoVs), each from a distinct wild-ruminant species in Ohio: sambar deer (Cervus unicolor), a waterbuck (Kobus ellipsiprymnus), a sable antelope (Hippotragus niger), and a white-tailed deer (Odocoileus virginianus). The fecal samples from the sambar deer, the waterbuck, and the white-tailed deer were collected during winter dysentery outbreaks and sporadic diarrhea cases in 1993 and 1994 (H. Tsunemitsu, Z. R. el-Kanawati, D. R. Smith, H. H. Reed, and L. J. Saif, J. Clin. Microbiol. 33:3264-3269, 1995). A fecal sample from a sable antelope was collected in 2003 from an Ohio wild-animal habitat during the same outbreak when a bovine-like CoV from a giraffe (GiCoV) was isolated (M. Hasoksuz, K. Alekseev, A. Vlasova, X. Zhang, D. Spiro, R. Halpin, S. Wang, E. Ghedin, and L. J. Saif, J. Virol. 81:4981-4990, 2007). For two of the CoVs (sambar deer and waterbuck), complete genomes from both the cell culture-adapted and gnotobiotic-calf-passaged strains were also sequenced and analyzed. Phylogenetically, wild-ruminant CoVs belong to group 2a CoVs, with the closest relatedness to recent bovine CoV (BCoV) strains. High nucleotide identities (99.4 to 99.6%) among the wild-ruminant strains and recent BCoV strains (BCoV-LUN and BCoV-ENT, isolated in 1998) further confirm the close relatedness. Comparative genetic analysis of CoVs of captive wild ruminants with BCoV strains suggests that no specific genomic markers are present that allow discrimination between the bovine strains and bovine-like CoVs from captive wild ruminants; furthermore, no specific genetic markers were identified that defined cell cultured or calf-passaged strains or the host origin of strains. The results of this study confirm prior reports of biologic and antigenic similarities between bovine and wild-ruminant CoVs and suggest that cattle may be reservoirs for CoVs that infect captive wild ruminants or vice versa and that these CoVs may represent host range variants of an ancestral CoV.     

Interaction of p190RhoGAP with C-terminal Domain of p120-catenin Modulates Endothelial Cytoskeleton and Permeability

p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820–843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation.     

Base-Calling Algorithm with Vocabulary (BCV) Method for Analyzing Population Sequencing Chromatograms

Sanger sequencing is a common method of reading DNA sequences. It is less expensive than high-throughput methods, and it is appropriate for numerous applications including molecular diagnostics. However, sequencing mixtures of similar DNA of pathogens with this method is challenging. This is important because most clinical samples contain such mixtures, rather than pure single strains. The traditional solution is to sequence selected clones of PCR products, a complicated, time-consuming, and expensive procedure. Here, we propose the base-calling with vocabulary (BCV) method that computationally deciphers Sanger chromatograms obtained from mixed DNA samples. The inputs to the BCV algorithm are a chromatogram and a dictionary of sequences that are similar to those we expect to obtain. We apply the base-calling function on a test dataset of chromatograms without ambiguous positions, as well as one with 3–14% sequence degeneracy. Furthermore, we use BCV to assemble a consensus sequence for an HIV genome fragment in a sample containing a mixture of viral DNA variants and to determine the positions of the indels. Finally, we detect drug-resistant Mycobacterium tuberculosis strains carrying frameshift mutations mixed with wild-type bacteria in the pncA gene, and roughly characterize bacterial communities in clinical samples by direct 16S rRNA sequencing.     

Energy Metabolic Changes in the Early Post-injury Period Following Traumatic Brain Injury in Rats

Impaired cerebral energy metabolism may be a major contributor to the secondary injury cascade that occurs following traumatic brain injury (TBI). To estimate the cortical energy metabolic state following mild and severe controlled cortical contusion (CCC) TBI in rats, ipsi-and contralateral cortical tissues were frozen in situ at 15 and 40 min post-injury and adenylate (ATP, ADP, AMP) levels were analyzed using high-performance liquid chromatography (HPLC) and the energy charge (EC) was calculated. At 15 min post-injury, mildly brain-injured animals showed a 43% decrease in cortical ATP levels and a 2.4-fold increase in AMP levels (P < 0.05), and there was a significant reduction of the ipsilateral cortical EC when compared to sham-injured animals (P < 0.05). At 40 min post-injury, the ipsilateral adenylate levels and EC had recovered to the values observed in the sham-injury group. In the severe CCC group, there was a 51% decrease in ipsilateral cortical ATP levels and a 5.3-fold increase in AMP levels with a significant reduction of cortical EC at 15 min post-injury (P < 0.05). At 40 min post-injury, a 2.6-fold ipsilateral increase in AMP levels and an 11% and 44% decrease in EC and ATP levels, respectively, remained (P < 0.05). A 37–38% reduction of the total adenylate pool was observed ipsilaterally in both CCC severity groups at the early time-point, and a 19% and 28% decrease remained in the mild and severe CCC groups, respectively, at 40 min post-injury. Significant contralateral ATP and EC changes were only observed in the severe CCC group at 40 min post-injury (P < 0.05). The energy-requiring secondary injury cascades that occur early post-injury do not challenge the brain tissue to the extent of ATP depletion and may provide a window of opportunity for therapeutic intervention.     

New records of the plant bug subfamilies Orthotylinae and Phylinae (Heteroptera,Miridae) from the European part of Russia

New records of plant bug species of the subfamilies Orthotylinae and Phylinae are given for selected regions of European Russia based on the extensive collections of the Zoological Institute of the Russian Academy of Sciences (St. Petersburg). The locality data and general distribution are presented for all the 97 species included in the list.