A protocol for the purification of bovine serum albumin free of deoxyribonuclease activity

Bovine serum albumin, free of deoxyribonuclease activity, was obtained in our laboratory using ion-exchange chromatography followed by acetylation. Chromatography on four different resins (DEAE-52, P-11, hydroxylapatite and Q Sepharose fast-flow) was examined. Fractions from Q Sepharose chromatography, eluted with a linear gradient 0-1.0 M NaCl and subsequently acetylated, proved to be the most effective method for obtaining deoxyribonuclease-free bovine serum albumin.     

Mechanisms of degradation of 2′-5′ oligoadenylates

We have studied the mechanisms of breakdown of 2'-5' oligoadenylates. We monitored the time-courses of degradation of ppp(A2'p5')nA (dimer to tetramer) and of 5'OH-(A2'p5')nA (dimer to pentamer) in unfractionated L1210 cell extract. The 5' triphosphorylated 2'-5' oligoadenylates are converted by a phosphatase activity. However, 2'-5' oligoadenylates are degraded mainly by phosphodiesterase activity which splits the 2'-5' phosphodiester bond sequentially at the 2' end to yield 5' AMP and one-unit-shorter oligomers. The nonlinear least-squares curve-fitting program CONSAM was used to fit these kinetics and to determine the degradation rate constant of each oligomer. Trimers and tetramers, whether 5' triphosphorylated or not, are degraded at the same rate, whereas 5' triphosphorylated dimer is rapidly hydrolyzed and 5'-OH dimer is the most stable oligomer. The interaction between degradation enzymes and the substrate strongly depends on the presence of a 5' phosphate group in the vicinity of the phosphodiester bond to be hydrolyzed; indeed, when this 5' phosphate group is present, as in pp/pA2'p5'A/or A2'/p5'A2'p5'A/, affinity is high and maximal velocity is low. Such a degradation pattern can control the concentration of 2'-5' oligoadenylates active on RNAse L either by limiting their synthesis (5' triphosphorylated dimer is the primer necessary for the formation of longer oligomers) and/or by converting them into inhibitory (e.g., monophosphorylated trimer) or inactive (e.g., nonphosphorylated oligomers) molecules.     

Growth modulation by epidermal growth factor (EGF) in human colonic carcinoma cells: constitutive expression of the human EGF gene

The functional role of epidermal growth factor (EGF) in epithelium-derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor-responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor-alpha (TGF-alpha) secretion. Growth stimulation and down-modulation of both high and low affinity EGF receptors were observed in the EGF-transfected Moser clones. Results of experiments using anti-EGF and anti-EGF-receptor antibody to block the proliferation of EGF-transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF-alpha were operative in these cells. By comparison, a growth-inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF-transfected KM12SM clones. Both the parental and EGF-transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti-EGF or anti-EGF-receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF-transfected KM12SM clones were non-autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium-derived human colonic carcinoma cells resulted in divergent altered growth characteristics.     

Recent Advances in Understanding Proteins Involved in Lipid Droplet Formation,Growth and Fusion

Lipid droplets （LDs） were once viewed as simple, inert lipid micelles. However, they are now known to be organelles with a rich proteome involved in a myriad of cellular processes. LDs are heterogeneous in nature with different sizes and compositions of phospholipids, neutral lipids and proteins. This review takes a focused look at the roles of proteins involved in the regulation of LD formation, expansion, and morphology. The related proteins are summarized such as the fat-specific protein （Fsp27）, fat storage-inducing trans- membrane （FIT） proteins, seipin and ADP-ribosylation factor 1-coat protein complex I （Arf-COPI）. Finally, we present important challenges in LD biology for a deeper understanding of this dynamic organelle to be achieved.     

The effects of vetrabutin chlorhydrate and oxytocin on stillbirth rate and asphyxia in swine

Oxytocin and vetrabutin chlorhydrate (VC) are used to reduce the duration of farrowing in swine. The objective of the present study was to evaluate the use of these products on intra-partum stillbirth (IPS) rate and asphyxia. At the onset of parturition, sows (n=180) were allocated to receive 2 mL of saline (control group), oxytocin (40 IU i.m.) or 100mg of VC per 60 kg of body weight, with all treatments given i.m. Oytocin-treated sows had a higher number of IPS than the VC and Control groups (means, 1.2, 0.8 and 0.6, respectively; P<0.001), and the highest percentage of ruptured umbilical cords (76.0, 9.4 and 37.5%; P<0.003). There were differences among groups for duration of farrowing (means, 163.0, 211.2 and 306.9 min in the oxytocin, VC and control groups; P<0.001), interval between piglets (13.9, 19.2 and 28.1 min; P<0.001), and in IPS, the incidence of ruptured umbilical cords was 76.0, 9.4 and 37.5% (P<0.003) and absence of a fetal heartbeat was 53.3, 16.9 and 12.5% (P<0.05). Although oxytocin decreased both duration of farrowing and interval between piglets by approximately 50% relative to control sows, it resulted in a significantly higher rate of IPS, in association with a much higher incidence of ruptured umbilical cord and absence of a fetal heartbeat. Treatment with VC reduced farrowing duration by approximately 1.5h, with an IPS rate that was not significantly different from controls but significantly lower than that of oxytocin-treated sows.     

Microfluidic‐Assisted Blade Coating of Compositional Libraries for Combinatorial Applications: The Case of Organic Photovoltaics

Microfluidic technologies are highly adept at generating controllable compositional gradients in fluids, a feature that has accelerated the understanding of the importance of chemical gradients in biological processes. That said, the development of versatile methods to generate controllable compositional gradients in the solid‐state has been far more elusive. The ability to produce such gradients would provide access to extensive compositional libraries, thus enabling the high‐throughput exploration of the parametric landscape of functional solids and devices in a resource‐, time‐, and cost‐efficient manner. Herein, the synergic integration of microfluidic technologies is reported with blade coating to enable the controlled formation of compositional lateral gradients in solution. Subsequently, the transformation of liquid‐based compositional gradients into solid‐state thin films using this method is demonstrated. To demonstrate efficacy of the approach, microfluidic‐assisted blade coating is used to optimize blending ratios in organic solar cells. Importantly, this novel technology can be easily extended to other solution processable systems that require the formation of solid‐state compositional lateral gradients.     

Legacies of Prehistoric Agricultural Practices Within Plant and Soil Properties Across an Arid Ecosystem

Closely integrated research between archaeologists and ecologists provides a long-term view of human land use that is rare in the ecological literature, allowing for investigation of activities that lead to enduring environmental outcomes. This extended temporal perspective is particularly important in aridlands where succession occurs slowly and ecosystem processes are mediated by abiotic, geomorphic factors. Numerous studies show that impacts from ancient human actions can persist, but few have explored the types of practices or mechanisms that lead to either transient or long-term environmental change. We compared plant and soil properties and processes from a range of landscape patch types in the Sonoran Desert of the US Southwest that supported different, well-documented prehistoric farming practices from AD 750–1300. Our results show that the types of ancient human activities that leave long-term ecological legacies in aridlands are those that fundamentally alter “slow variables” such as soil properties that regulate the timing and supply of water. Prehistoric Hohokam floodwater-irrigation practices, but not dryland farming techniques, substantially altered soil texture, which was strongly associated with desert plant community and functional composition. However, prehistoric agriculture did not consistently alter long-term nutrient availability and thus had no impact on “fast variables” such as production of seasonal annual plants that are restricted to periods of ample rainfall. In this arid ecosystem, the inverse texture model explained patterns in plant functional composition at large scales, but is less predictive of production of short-lived desert annuals that experience a more mesic precipitation regime.     

Huntington's Disease Induced Cardiac Amyloidosis Is Reversed by Modulating Protein Folding and Oxidative Stress Pathways in the Drosophila Heart

Amyloid-like inclusions have been associated with Huntington''s disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of contractile proteins, leads to mitochondrial dysfunction and increases oxidative stress in cardiomyocytes leading to abnormal cardiac function. We conclude that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients.     

Publication of descriptions of novel bacterial taxa in <Emphasis Type="Italic">Antonie van Leeuwenhoek</Emphasis>

The species from the order Neisseriales are currently distinguished from other bacteria on the basis of branching in 16S rRNA gene trees. For this order containing a single family, Neisseriaceae, no distinctive molecular, biochemical, or phenotypic characters are presently known. We report here detailed phylogenetic and comparative analyses on the 27 genome sequenced species of the order Neisseriales. Our comparative genomic analyses have identified 54 conserved signature indels (CSIs) in widely distributed proteins that are specific for either all of the sequenced Neisseriales species or a number of clades within this order that are also supported by phylogenetic analyses. Of these CSIs, 11 are specifically present in all of the sequenced species from this order, but are not found in homologous proteins from any other bacteria. These CSIs provide novel molecular markers specific for, and delimiting, this order. Twenty-one CSIs in diverse proteins are specific for a group comprised of the genera Neisseria, Eikenella, Kingella, and Simonsiella (Clade I), which are obligate host-associated organisms, lacking flagella and exhibiting varied morphology. The species from these genera also formed a strongly supported clade in phylogenetic trees based upon concatenated protein sequences; a monophyletic grouping of these genera and other genera displaying similar morphological characteristics was also observed in the 16S rRNA gene tree. A second clade (Clade II), supported by seven of the identified CSIs and phylogenetic trees based upon concatenated protein sequences, grouped together species from the genera Chromobacterium, Laribacter, and Pseudogulbenkiania that are rod-shaped bacteria, which display flagella-based motility and are capable of free living. The remainder of the CSIs were uniquely shared by smaller groups within these two main clades. Our analyses also provide novel insights into the evolutionary history of the Neisseriales and suggest that the CSIs that are specific for the Clade I species may play an important role in the evolution of obligate host-association within this order. On the basis of phylogenetic analysis, the identified CSIs, and conserved phenotypic characteristics of different Neisseriales genera, we propose a division of this order into two families: an emended family Neisseriaceae (corresponding to Clade I) containing the genera Alysiella, Bergeriella, Conchiformibius, Eikenella, Kingella, Neisseria, Simonsiella, Stenoxybacter, Uruburuella and Vitreoscilla and a new family, Chromobacteriaceae fam. nov., harboring the remainder of the genera from this order (viz. Andreprevotia, Aquaspirillum, Aquitalea, Chitinibacter, Chitinilyticum, Chitiniphilus, Chromobacterium, Deefgea, Formivibrio, Gulbenkiania, Iodobacter, Jeongeupia, Laribacter, Leeia, Microvirgula, Paludibacterium, Pseudogulbenkiania, Silvimonas, and Vogesella).