Mutation of an upstream cleavage site in the BMP4 prodomain leads to tissue-specific loss of activity

ProBMP4 is initially cleaved at a site adjacent to the mature ligand (the S1 site) allowing for subsequent cleavage at an upstream (S2) site. Mature BMP4 synthesized from a precursor in which the S2 site cannot be cleaved remains in a complex with the prodomain that is targeted for lysosomal degradation, and is thus less active when overexpressed in Xenopus. Here we report that mice carrying a point mutation that prevents S2 processing show severe loss of BMP4 activity in some tissues, such as testes and germ cells, whereas other tissues that are sensitive to Bmp4 dosage, such as the limb, dorsal vertebrae and kidney, develop normally. In a haploinsufficient background, inability to cleave the S2 site leads to embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, eye and heart. These data demonstrate that cleavage of the S2 site is essential for normal development and, more importantly, suggest that this site might be selectively cleaved in a tissue-specific fashion. In addition, these studies provide the first genetic evidence that BMP4 is required for dorsal vertebral fusion and closure of the ventral body wall.     

Differential regulation of proteasome activity in the nucleus and the synaptic terminals

Proteasome is a multi-subunit proteolytic complex that degrades proteins covalently linked to multiple molecules of ubiquitin. Earlier studies showed a role for the ubiquitin-proteasome pathway in several models of long-term memory and other forms of synaptic plasticity. In Aplysia, the ubiquitin-proteasome pathway has been shown to contribute to the induction of long-term facilitation. In other model systems, ubiquitin-proteasome-mediated proteolysis has also been shown to play a role in synapse development. Previous studies of synaptic plasticity focused on changes in components or the substrates of the ubiquitin-proteasome pathway in whole neurons. Modification of specific synapses would require precise spatial and temporal regulation of the components of the ubiquitin-proteasome pathway within the subcellular compartments of neurons during learning. As a first step towards testing the idea of local regulation of the ubiquitin-proteasome pathway in neurons, we investigated proteasome activity in nuclear and synaptosomal fractions. Here we show that proteasome activity in the synaptic terminals is higher compared to the activity in the nucleus in the Aplysia nervous system as well as in the mouse brain. Furthermore, the proteasome activity in the two neuronal compartments is differentially modulated by protein kinases. Differential regulation of proteasome activity in neuronal compartments such as the synaptic terminals is likely to be a key mechanism underlying synapse-specific plasticity.     

Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.     

The role of BiP in endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain induced by cytomegalovirus proteins

Human cytomegalovirus (HCMV1) US11 and US2 proteins cause rapid degradation of major histocompatibility complex (MHC) molecules, apparently by ligating cellular endoplasmic reticulum (ER)-associated degradation machinery. Here, we show that US11 and US2 bind the ER chaperone BiP. Four related HCMV proteins, US3, US7, US9, and US10, which do not promote degradation of MHC proteins, did not bind BiP. Silencing BiP reduced US11- and US2-mediated degradation of MHC class I heavy chain (HC) without altering the synthesis or translocation of HC into the ER or the stability of HC in the absence of US11 or US2. Induction of the unfolded protein response (UPR) did not affect US11-mediated HC degradation and could not explain the stabilization of HC when BiP was silenced. Unlike in yeast, BiP did not act by maintaining substrates in a retrotranslocation-competent form. Our studies go beyond previous observations in mammalian cells correlating BiP release with degradation, demonstrating that BiP is functionally required for US2- and US11-mediated HC degradation. Further, US2 and US11 bound BiP even when HC was absent and degradation of US2 depended on HC. These data were consistent with a model in which US2 and US11 bridge HC onto BiP promoting interactions with other ER-associated degradation proteins.     

The evolution of California's wild radish has resulted in the extinction of its progenitors

If two previously isolated taxa mutually assimilate through hybridization and subsequent biparental introgression, and if their introgressed descendants have the same or higher fitness than their parents, then gene flow should result in the local extinction of parental taxa via replacement by hybrid derivatives. These dramatic events may occur rapidly, even in a few generations. Given the speed at which such extinction by hybridization may occur, it may be difficult to identify that the process has occurred. Thus, documented instances of extinction by hybridization are rare, and especially so for cases in which both parents are replaced by the hybrid lineage. Here we report morphological and allozyme evidence for the local extinction of two Raphanus species in California via replacement by their hybrid-derived descendants. The results from a greenhouse experiment demonstrate that California wild radishes have a specific combination of traits from their progenitors, and comparison of our results to that of an earlier report indicate that pure parental types are no longer present in the wild. Our results also show the hybrid-derived lineage has transgressive fruit weight compared to its parents. Allozyme analysis demonstrates that California wild radishes are derived from hybridization between the putative parental species. However, that analysis also demonstrates that California wild radish has now become an evolutionary entity separate from both of its parents. We suggest that the aggressive colonizing behavior of the hybrid-derived lineage probably results from a novel combination of parental traits, rather than genetic variability of the population per se.     

A novel calcium-independent peripheral membrane-bound form of annexin B12

Annexins are soluble proteins that can interact with membranes in a Ca2+-dependent manner. Recent studies have shown that they can also undergo Ca2+-independent membrane interactions that are modulated by pH and phospholipid composition. Here, we investigated the structural changes that occurred during Ca2+-independent interaction of annexin B12 with phospholipid vesicles as a function of pH. Electron paramagnetic resonance analysis of a helical hairpin encompassing the D and E helices in the second repeat of the protein showed that this region refolded and formed a continuous amphipathic alpha helix following Ca2+-independent binding to membranes at mildly acidic pH. At pH 4.0, this helix assumed a transmembrane topography, but at pH approximately 5.0-5.5, it was peripheral and approximately parallel to the membrane. The peripheral form was reversibly converted into the transmembrane form by lowering the pH and vice versa. Furthermore, analysis of vesicles incubated with annexin B12 using freeze-fracture electron microscopy methods showed classical intramembrane particles at pH 4.0 but none at pH 5.3. Together, these data raise the possibility that the peripheral-bound form of annexin B12 could act as a kinetic intermediate in the formation of the transmembrane form of the protein.     

Pentapeptide repeat proteins

The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.     

Callus culture and an unconventional pattern of sporophyte regeneration in Drynaria quercifolia—A medicinal fern

Summary Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop’s (1865) medium supplemented with 20 gl⁻¹ sucrose, 8gl⁻¹ agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mgl⁻¹ 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mgl⁻¹ 6-(δ, δ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.     

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.     

Computer modeling of nitroxide spin labels on proteins

Electron paramagnetic resonance using site‐directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron–electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F‐BAR), endophilin, and α‐synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r² = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 35–44, 2012.