HPLC‐Fluorescence Method for the Enantioselective Analysis of Propranolol in Rat Serum Using Immobilized Polysaccharide‐Based Chiral Stationary Phase

A stereoselective high‐performance liquid chromatographic (HPLC) method was developed and validated to determine S‐(?)‐ and R‐(+)‐propranolol in rat serum. Enantiomeric resolution was achieved on cellulose tris(3,5‐dimethylphenylcarbamate) immobilized onto spherical porous silica chiral stationary phase (CSP) known as Chiralpak IB. A simple analytical method was validated using a mobile phase consisted of n‐hexane‐ethanol‐triethylamine (95:5:0.4%, v/v/v) at a flow rate of 0.6 mL min^‐1 and fluorescence detection set at excitation/emission wavelengths 290/375 nm. The calibration curves were linear over the range of 10–400 ng mL^‐1 (R = 0.999) for each enantiomer with a detection limit of 3 ng mL^‐1. The proposed method was validated in compliance with ICH guidelines in terms of linearity, accuracy, precision, limits of detection and quantitation, and other aspects of analytical validation. Actual quantification could be made for propranolol isomers in serum obtained from rats that had been intraperitoneally (i.p.) administered a single dose of the drug. The proposed method established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology. Molecular modeling studies including energy minimization and docking studies were first performed to illustrate the mechanism by which the active enantiomer binds to the β‐adrenergic receptor and second to find a suitable interpretation of how both enantiomers are interacting with cellulose tris(3,5‐dimethylphenylcarbamate) CSP during the process of resolution. The latter interaction was demonstrated by calculating the binding affinities and interaction distances between propranolol enantiomers and chiral selector. Chirality 26:194–199, 2014. © 2014 Wiley Periodicals, Inc.     

Nonlinear relationships among evolutionary rates identify regions of functional divergence in heat-shock protein 70 genes

The neutral theory predicts that, in comparisons among related genes, the number of amino acid replacements per site in a given gene region should be a linear function of that in another region of the same gene, unless the genes have diverged functionally in one region. Therefore, nonlinearity of this relationship can be used to identify regions of possible functional divergence among members of a multigene family. This method of analysis was applied to members of the heat-shock protein 70 (HSP70) gene family, which encode highly conserved ATP- dependent chaperone proteins found in all organisms. A nonlinear relationship was found between the rate of amino acid replacement in the conserved IA domain of the ATPase portion of the molecule and that in other ATPase domains and the peptide-binding domain. These results suggest that genes in the HSP70 subfamily C (dnaK of bacteria and SSC1 of yeast) may have diverged functionally from other subfamilies in the ATPase domains, especially IIB, whereas SSB1 of yeast has diverged markedly in the peptide-binding domain. Functional divergence within these regions is consistent with what is known about functional differences between the HSP70 subfamilies in yeast.      

Directional and balancing selection in human beta-defensins

Background  

In primates, infection is an important force driving gene evolution, and this is reflected in the importance of infectious disease in human morbidity today. The beta-defensins are key components of the innate immune system, with antimicrobial and cell signalling roles, but also reproductive functions. Here we examine evolution of beta-defensins in catarrhine primates and variation within different human populations.     

Drivers of beta diversity of macroinvertebrate communities in tropical forest streams

1. There has recently been increasing interest in patterns of beta diversity but we still lack a comprehensive understanding of these patterns in various regions (e.g. the tropics), ecosystems (e.g. streams) and organism groups (e.g. invertebrates). 2. Our aim was to investigate the patterns of beta diversity of stream macroinvertebrates in relation to key environmental (i.e. stream size, pH and habitat degradation) and geographical variables (i.e. latitude, longitude, altitude) in a tropical region. We surveyed a total of 8–10 riffle sites in each of 34 streams (altogether 337 riffle sites were sampled) in Peninsular Malaysia to examine variation in macroinvertebrate community composition at within‐stream and among‐stream scales. 3. Based on test of homogeneity of dispersion, we found that the streams studied differed significantly in within‐stream variation in community composition (i.e. among‐site variation of within stream beta diversity). The patterns were similar based on Bray–Curtis coefficient on abundance data, Sorensen coefficient on presence–absence data and Simpson coefficient on presence–absence data. We also found that within‐stream beta diversity was significantly related to stream size, pH and latitude, with each of these variables individually accounting for around 20% of the variation in beta diversity in simple regressions, while the total variation explained by the three significant variables amounted to around 50% in multiple regressions. By contrast, habitat degradation, longitude and altitude were not significantly related to beta diversity. We also found that the factor drainage basin accounted for much of the variation in beta diversity in general linear models, suppressing the effects of environmental variables. 4. We concluded that within‐stream beta diversity is mainly related to a combination of the identity of a drainage basin and stream environmental factors. Our findings provide important background for stream environmental assessment and conservation planning by emphasising that (i) macroinvertebrate communities within streams are not homogeneous, but show considerable beta diversity, (ii) streams differ in their degree of within‐stream beta diversity, (iii) stream size and water pH should be considered in applied contexts related to within‐stream beta diversity and (iv) historical effects may be different in different drainage basins and may affect present‐day patterns of within‐stream beta diversity.     

Blebs produced by actin–myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs

Apoptosis is a fundamental homeostatic mechanism essential for the normal growth, development and maintenance of every tissue and organ. Dying cells have been defined as apoptotic by distinguishing features, including cell contraction, nuclear fragmentation, blebbing, apoptotic body formation and maintenance of intact cellular membranes to prevent massive protein release and consequent inflammation. We now show that during early apoptosis limited membrane permeabilization occurs in blebs and apoptotic bodies, which allows release of proteins that may affect the proximal microenvironment before the catastrophic loss of membrane integrity during secondary necrosis. Blebbing, apoptotic body formation and protein release during early apoptosis are dependent on ROCK and myosin ATPase activity to drive actomyosin contraction. We identified 231 proteins released from actomyosin contraction-dependent blebs and apoptotic bodies by adapted SILAC (stable isotope labeling with amino acids in cell culture) combined with mass spectrometry analysis. The most enriched proteins released were the nucleosomal histones, which have previously been identified as damage-associated molecular pattern proteins (DAMPs) that can initiate sterile inflammatory responses. These results indicate that limited membrane permeabilization occurs in blebs and apoptotic bodies before secondary necrosis, leading to acute and localized release of immunomodulatory proteins during the early phase of active apoptotic membrane blebbing. Therefore, the shift from apoptosis to secondary necrosis is more graded than a simple binary switch, with the membrane permeabilization of apoptotic bodies and consequent limited release of DAMPs contributing to the transition between these states.     

Phylogeny of Six Genera of the Subclass Haptoria (Ciliophora,Litostomatea) Inferred from Sequences of the Gene Coding for Small Subunit Ribosomal RNA

ABSTRACT. The small subunit ribosomal RNA genes of nine species belonging to six genera of litostome ciliates, namely Amphileptus aeschtae, Chaenea teres, Chaenea vorax, Lacrymaria marina, Litonotus paracygnus, Loxophyllum sp.‐GD‐070419, Loxophyllum jini, Loxophyllum rostratum, and Phialina salinarum, were sequenced for the first time. Phylogenetic trees were constructed using different methods to assess the inter‐ and intra‐generic relationships of haptorians, of which Chaenea, Lacrymaria, Litonotus, and Phialina were analyzed for the first time based on molecular data. Monophyly of the order Pleurostomatida was strongly confirmed, and the two existing families of pleurostomatids, created on the basis of morphology, were confirmed by molecular evidence. Within the Pleurostomatida, Siroloxophyllum utriculariae occupied a well‐supported position basal to the Loxophyllum clade, supporting the separation of these genera from one another. Both the subclass Haptoria and the order Haptorida were partially unresolved, possibly paraphyletic assemblages of taxa in all analyses, creating doubts about the traditional placement of some haptorid taxa. The existing sequence of L. rostratum in GenBank (DQ411864) was conspicuously different from that of the isolate from Qingdao, China sequenced in the present work, indicating that they are different species. The isolate from Qingdao was verified as L. rostratum by morphological analysis, and the published morphology of existing GenBank record of L. rostratum is different from it. Based on both morphological and molecular evidence, the latter may be congeneric with an undescribed species of Loxophyllum from Guangdong Province, China.     

Re‐targeting of a plant defense protease by a cyst nematode effector

Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector‐coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two‐hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain‐like cysteine protease (PLCP) ‘Responsive to Dehydration 21A’ (RD21A), which has been shown to function in the plant defense response. Activity‐based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re‐localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two‐hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector‐mediated re‐localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense‐inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.     

The Detection of Inhibitors of the Sclerotinia sclerotiorum (Lib.) de Bary Extracellular Proteinases in Sunflower

Abstract: Water-soluble protein fractions from leaves, seeds and heads of sunflower were shown to contain inhibitors of trypsin, chymotrypsin and extracellular proteinases from Sclerotinia sclerotiorum , a pathogen of sunflower, and Colletotrichum lindemuthianum. These included bifunctional inhibitors of trypsin and subtilisin. Comparison with the patterns of inhibition of standard proteinases indicated that the major extracellular proteinases of S. sclerotiorum are subtilisin-like. It is speculated that the sunflower inhibitors play a role in conferring resistance to fungal infection.