High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

Soil and greenhouse gas responses to biochar additions in a temperate hardwood forest

Biochar additions can improve soil fertility and sequester carbon, but biochar effects have been investigated primarily in agricultural systems. Biochar from spruce and maple sawdust feedstocks (with and without inorganic phosphorus in a factorial design) were added to plots in a commercially managed temperate hardwood forest stand in central Ontario, Canada; treatments were applied as a top‐dressing immediately prior to fall leaf abscission in September 2011. Forests in this region have acidic, sandy soils, and due to nitrogen deposition may exhibit phosphorus, calcium, and magnesium limitation. To investigate short‐term impacts of biochar application on soil nutrient supply and greenhouse gas fluxes as compared to phosphorus fertilization, data were collected over the first year after treatment application; linear mixed models were used to analyze data. Two to six weeks after treatment application, there were higher concentrations of potassium in spruce and maple biochar plots, and phosphorus in spruce biochar plots, as compared to the control treatment. There were higher concentrations of calcium, magnesium, and phosphorus in the phosphorus plots. In the following spring and summer (9–12 months after treatment application), there were higher soil calcium concentrations in maple biochar plots, and phosphorus plots still had higher soil phosphorus concentrations than control plots. No treatment effects on fluxes of carbon dioxide, methane, or nitrous oxide were detected in the field; however, laboratory incubations after 12 months showed higher microbial respiration in soils from maple biochar plots as compared to spruce biochar, despite no effect on microbial biomass. The results suggest that the most important short‐term impact of biochar additions in this system is the increased supply of the limiting plant nutrients phosphorus and calcium. We expect that larger changes in mineral soil physical and chemical properties will occur when the surface‐applied biochar becomes incorporated into the soil after a few years.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Inactivation of Influenza A Virus on Copper versus Stainless Steel Surfaces

Influenza A virus particles (2 × 10⁶) were inoculated onto copper or stainless steel and incubated at 22°C at 50 to 60% relative humidity. Infectivity of survivors was determined by utilizing a defined monolayer with fluorescent microscopy analysis. After incubation for 24 h on stainless steel, 500,000 virus particles were still infectious. After incubation for 6 h on copper, only 500 particles were active.     

Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects

Cloned EcR and USP cDNAs encoding the ecdysone receptors of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were manipulated to allow the co-expression of their ligand binding domains (LBDs) in insect cells using a baculovirus vector. Recombinant DE/F segment pairs (and additionally, for H. armigera, an E/F segment pair) from the EcR and USP proteins associated spontaneously with high affinity to form heterodimers that avidly bound an ecdysteroid ligand. This shows that neither ligand nor D-regions are essential for the formation of tightly associated and functional LBD heterodimers. Expression levels ranged up to 16.6mg of functional apo-LBD (i.e., unliganded LBD) heterodimer per liter of recombinant insect cell culture. Each recombinant heterodimer was affinity-purified via an oligo-histidine tag at the N-terminus of the EcR subunit, and could be purified further by ion exchange and/or gel filtration chromatography. The apo-LBD heterodimers appeared to be more easily inactivated than their ligand-containing counterparts: after purification, populations of the former were <40% active, whereas for the latter >70% could be obtained as the ligand-LBD heterodimer complex. Interestingly, we found that the amount of ligand bound by recombinant LBD heterodimer preparations could be enhanced by the non-denaturing detergent CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate). Purity, integrity, size and charge data are reported for the recombinant proteins under native and denaturing conditions. Certain intra- and intermolecular disulfide bonds were observed to form in the absence of reducing agents, and thiol-specific alkylation was shown to suppress this phenomenon but to introduce microheterogeneity.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.     

Use of copper cast alloys to control Escherichia coli O157 cross-contamination during food processing

The most notable method of infection from Escherichia coli O157 (E. coli O157) is through contaminated food products, usually ground beef. The objective of this study was to evaluate seven cast copper alloys (61 to 95% Cu) for their ability to reduce the viability of E. coli O157, mixed with or without ground beef juice, and to compare these results to those for stainless steel. E. coli O157 (NCTC 12900) (2 x 10(7) CFU) mixed with extracted beef juice (25%) was inoculated onto coupons of each copper cast alloy or stainless steel and incubated at either 22 degrees C or 4 degrees C for up to 6 h. E. coli O157 viability was determined by plate counts in addition to staining in situ with the respiratory indicator fluorochrome 5-cyano-2,3-ditolyl tetrazolium. Without beef extract, three alloys completely killed the inoculum during the 6-h exposure at 22 degrees C. At 4 degrees C, only the high-copper alloys (>85%) significantly reduced the numbers of O157. With beef juice, only one alloy (95% Cu) completely killed the inoculum at 22 degrees C. For stainless steel, no significant reduction in cell numbers occurred. At 4 degrees C, only alloys C83300 (93% Cu) and C87300 (95% Cu) significantly reduced the numbers of E. coli O157, with 1.5- and 5-log kills, respectively. Reducing the inoculum to 10(3) CFU resulted in a complete kill for all seven cast copper alloys in 20 min or less at 22 degrees C. These results clearly demonstrate the antimicrobial properties of cast copper alloys with regard to E. coli O157, and consequently these alloys have the potential to aid in food safety.     

Nectin 4 is the epithelial cell receptor for measles virus

Measles virus (MV) causes acute respiratory disease, infects lymphocytes and multiple organs, and produces immune suppression leading to secondary infections. In rare instances it can also cause persistent infections in the brain and central nervous system. Vaccine and laboratory-adapted strains of MV use CD46 as a receptor, whereas wild-type strains of MV (wtMV) cannot. Both vaccine and wtMV strains infect lymphocytes, monocytes, and dendritic cells (DCs) using the signaling lymphocyte activation molecule (CD150/SLAM). In addition, MV can infect the airway epithelial cells of the host. Nectin 4 (PVRL4) was recently identified as the epithelial cell receptor for MV. Coupled with recent observations made in MV-infected macaques, this discovery has led to a new paradigm for how the virus accesses the respiratory tract and exits the host. Nectin 4 is also a tumor cell marker which is highly expressed on the apical surface of many adenocarcinoma cell lines, making it a potential target for MV oncolytic therapy.