Phylogeny of the Drosophila saltans species group based on combined analysis of nuclear and mitochondrial DNA sequences

Nucleotide sequences from two nuclear loci, alcohol dehydrogenase and internal transcribed spacer-1 of the nuclear ribosomal DNA repeats, and two mitochondrial genes, cytochrome oxidase I and cytochrome oxidase II, were determined from nine species in the Drosophila saltans species group. The partition homogeneity test and partitioned Bremer support were used to measure incongruence between phylogenetic hypotheses generated from individual partitions. Individual loci were generally congruent with each other and consistent with the previously proposed morphological hypothesis, although they differed in level of resolution. Since extreme conflict between partitions did not exist, the data were combined and analyzed simultaneously. The total evidence method gave a more resolved and highly supported phylogeny, as indicated by bootstrap proportions and decay indices, than did any of the individual analyses. The cordata and elliptica subgroups, considered to have diverged early in the history of the D. saltans group, were sister taxa to the remainder of the saltans group. The sturtevanti subgroup, represented by D. milleri and D. sturtevanti, occupies an intermediate position in this phylogeny. The saltans and parasaltans subgroups are sister clades and occupy the most recently derived portion of the phylogeny. As with previous morphological studies, phylogenetic relationships within the saltans subgroup were not satisfactorily resolved by the molecular data.      

Protein extraction by reverse micelles: studies on the recovery of horseradish peroxidase

Phase transfer studies were carried out on the solubilization of horseradish peroxidase (HRP) (E.C. 1.11.1.7) in reverse micelles formed in isooctane using the anionic surfactant, aerosol OT, at concentrations between 50 and 110mM. The selectivity of this methodology was tested, because the HRP used comprised a mixture of seven different isoenzymes with a wide range of isoelectric points. Forward and backward transfers were carried out in wellstirred vessels until equilibrium was reached. Significant protein partitioning could only be obtained by using NaCl to adjust ionic strength in pH range between 1.5 and 3.5, with a maximum at pH 3. The back transfer process was best at pH 8 with 80mM phosphate buffer and 1 M KCI. A loss of 1% to 3% of the surfactant through precipitation at the interface at pH<4 was observed, which may be due to instability in this pH region, because, even without protein, a similar precipitate was noticed. Protein partitioning was approximately constant when the ionic strength was increased up to 1 MNaCl at pH 3, but protein recovery in back transfer decreased accordingly. Hydrophobic interactions together with association between the protein and surfactant might be responsible for that behavior. Protein partitioning remained the same when the surfactant concentration was decreased to 50 mM, at the expense of higher variability. HPLC chromatograms showed no apparent damage to the protein after reverse micellar extraction. Protein partitioning is best when the temperature is kept at 25xC. The amount of protein and specific activity recovered strongly depends on the phase ratio used during forward transfer. Overall activity recovery varied from 87% to 136% when the phase ratio was increased from 1:1 to 30:1 in forward transfer. This behavior may be due to a change in the ratio of the three isoenzymes recovered after the backward transfer process, with the most active one being increasingly enriched at higher phase ratios. (c) 1994 John Wiley & Sons, Inc.     

Phylogenetic analysis supports horizontal transfer of P transposable elements

Nucleotide sequence comparisons were used to investigate the evolution of P transposable elements and the possibility that horizontal transfer has played a role in their occurrence in natural populations of Drosophila and other Diptera. The phylogeny of P elements was examined using published sequences from eight dipteran taxa and a new, partial sequence from Scaptomyza elmoi. The results from a number of different analyses are highly consistent and reveal a P-element phylogeny that contradicts the phylogeny of the species. At least three instances of horizontal transfer are necessary to explain this incongruence, but other explanations cannot be ruled out at this time.      

Structural phosphoprotein M2-1 of the human respiratory syncytial virus is an RNA binding protein

The structural phosphoprotein M2-1 of human respiratory syncytial virus (HRSV) Long strain shows RNA binding capacity in three different assays that detect RNA-protein complexes: cross-linking, gel retardation, and Northern-Western assays. It is able to bind HRSV leader RNA specifically with cooperative kinetics, with an apparent K(d) of at least 90 nM. It also binds to long RNAs with no sequence specificity. The RNA binding domain has been located between amino acid residues 59 and 85, at the NH(2) terminus of the protein. This region contains the phosphorylatable amino acid residues threonine 56 and serine 58, whose modification decreases the binding capacity of M2-1 protein to long RNAs.     

Differences in growth and water relations among Phaseolus vulgaris cultivars in response to induced drought stress

Relatively little ecophysiological research has been conducted to determine the responses to drought of Phaseolus vulgaris. Four bean cultivars (cvs.) from Brazil, A320, Carioca, Ouro Negro and Xodó were submitted to an imposed water deficit in order to evaluate the importance of some adaptive mechanisms of drought resistance through the analysis of growth parameters, water status, gas exchange and indicators of tolerance mechanisms at the cellular level. During the drought treatment, relative growth rates were more reduced for A320 and Xodó than Carioca and Ouro Negro. A320 closed its stomata very rapidly and complete stomatal closure was obtained at Psi(w)=-0.6 MPa, in contrast to the other cvs. where stomata were fully closed only at Psi(w)=-0.9 MPa. Net assimilation rates were closely related to stomatal conductances. Mechanisms at the cellular level appeared to be mostly important for higher tolerance. Carioca and Ouro Negro, when compared to A320 and Xodó, were characterized by having better drought tolerance mechanisms and higher tissue water retention capacity leading to a better growth under water deficits. The leaf dehydration rates of those cvs. were slow whereas those of the drought sensitive cvs. were rapid. The results were confirmed by the electrolyte leakage test and leaf osmotic potential measurements, which indicated higher membrane resistance and osmotic adjustment in the two tolerant cvs. Carioca and Ouro Negro. It appears from this study that despite being cultivated in the same geographical region, the four cvs. of P. vulgaris displayed somewhat different drought adaptive capacities for prolonged drought during the vegetative phase.     

Optimal process synthesis for the production of multiple recombinant proteins

This paper presents a novel solution strategy for the synthesis of multiproduct and multihost protein production processes. There are several possible hosts that may express each of the products, and different downstream processing separation and purification tasks are needed, which in part depend on the host selection. Moreover, alternative unit operations may be available for some of these separation tasks. Finally, these processing units may be arranged in different configurations. A single mixed-integer optimization model represents the different decisions involved in synthesizing a plant for producing multiple proteins. The mathematical model optimizes the profit of the multiproduct plant and allows the decisions to be made simultaneously, namely, the choice of hosts, downstream operations, the configuration and size of units, as well as their scheduling. An example is solved for a plant that must produce four proteins for which there are alternative hosts for their expression (Escherichia coli, Chinese hamster ovary cells, and yeast that, depending on the product, may express it as an extracellular or intracellular protein) that require 15 stages with choices of unit operations as well as in or out of phase operations. Given the very large quantity of novel recombinant proteins for a number of novel therapeutic uses presently being approved or "in the pipeline", multiproduct and multihost recombinant protein production plants have recently been or are being built for the manufacture of these products. The strategy presented in this paper is of crucial value for the optimal utilization of such plants.     

Obtaining a protein concentrate from integral defatted sunflower flour

Proteins which are found in integral defatted sunflower flour (27% of protein in dry weight) allow us to produce a protein concentrate by means of extraction of proteins with a basic pH solution, followed by their precipitation with an acid pH solution. Once the suitable conditions for pH and temperature were fixed in order to carry out these processes, a solid proteic concentrate (71% of protein in dry weight) was obtained which was rich in glutamic and aspartic acids, with a liquid supernatant very rich in phosphorus and potassium, which might be used as an agricultural fertilizer.     

Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:307–315, 2015     

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

Aminopyrimidines and Derivatives. 20. On the Acetylations of 5-Amino-4-Glycosylmino Pyrimidines

Abstract

The acetylation of 5-amino-4-glycosylamino pyrimidines afforded a wide variety of compounds, depending on the reaction conditions. A systematic study has been carried out and some conclusions with synthetic utility are proposed.