The timed-pregnant baboon animal model can be used for determining the role of soluble vascular endothelial growth factor receptors 1 and 2 during development

BACKGROUND: During pregnancy, mechanisms that allow for regulation of continuous fetal and placental vasculogenesis with prevention of maternal neo-vascularization remain elusive. The vascular endothelial growth factor (VEGF) biological system has a key role during vasculogenesis. The aims of this study were to validate a bioassay for soluble vascular endothelial growth factor receptors 1 and 2 (sVEGFR-1 and sVEGFR-2) in baboon plasma and to determine the maternal and fetal plasma concentration of these receptors at the end of the baboon pregnancy. METHODS: Maternal peripheral blood samples were obtained from eight baboons (Papio anubis) prior to elective cesarean section and from the umbilical cord after the fetuses were delivered. Spike and recovery experiments at various concentrations in pooled baboon maternal plasma were used to validate a human quantitative sandwich immunoassay for sVEGFR-1 and -2. Concentrations of sVEGFR-1 and -2 were then determined in maternal and fetal plasma samples. RESULTS: No significant correlations were observed between sVEGFR-1 or -2 concentrations in maternal and fetal circulations. The concentration of sVEGFR-1 was at least 30 times greater and that of sVEGFR-2 approximately two times greater, in maternal than in cord plasma (both P < 0.01). CONCLUSION: These findings suggest that baboons can be used to study the regulation of vasculogenesis during pregnancy.     

Evaluation of plastic composite-supports for enhanced ethanol production in biofilm reactors

Biofilms are a natural form of cell immobilization that result from microbial attachment to solid supports. Biofilm reactors with polypropylene composite-supports containing up to 25% (w/w) of various agricultural materials (corn hulls, cellulose, oat hulls, soybean hulls or starch) and nutrients (soybean flour or zein) were used for ethanol production. Pure cultures ofZymomonas mobilis, ATCC 31821 orSaccharomyces cerevisiae ATCC 24859 and mixed cultures with either of these ethanol-producing microorganisms and the biofilm-formingStreptomyces viridosporus T7A ATCC 39115 were evaluated. An ethanol productivity of 374g L^–1 h^–1 (44% yield) was obtained on polypropylene composite-supports of soybean hull-zein-polypropylene by usingZ. mobilis, whereas mixed-culture fermentations withS. viridosporus resulted in ethanol productivity of 147.5 g L^–1 h^–1 when polypropylene composite-supports of corn starch-soybean flour were used. WithS. cerevisiae, maximum productivity of 40 g L^–1 h^–1 (47% yield) was obtained on polypropylene composite-supports of soybean hull-soybean flour, whereas mixed-culture fermentation withS. viridosporus resulted in ethanol productivity of 190g L^–1 h^–1 (35% yield) when polypropylene composite-supports of oat hull-polypropylene were used. The maximum productivities obtained without supports (suspension culture) were 124 g L^–1 h^–1 and 5 g L^–1 h^–1 withZ. mobilis andS. cerevisiae, respectively. Therefore, forZ. mobilis andS. cerevisiae, ethanol productivities in biofilm fermentations were three- and eight-fold higher than suspension culture fermentations, respectively. Biofilm formation on the chips was detected by weight change and Gram staining of the support material at the end of the fermentation. The ethanol production rate and concentrations were consistently greater in biofilm reactors than in suspension cultures.This is Journal Paper No. J-16356 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3253     

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.      

Detection of MET mRNA in gastric cancer in situ. Comparison with immunohistochemistry and sandwich immunoassays

Determination of predictive biomarkers by immunohistochemistry (IHC) relies on antibodies with high selectivity. RNA in situ hybridization (RNA ISH) may be used to confirm IHC and may potentially replace it if suitable antibodies are not available or are insufficiently selective to discriminate closely related protein isoforms. We validated RNA ISH as specificity control for IHC and as a potential alternative method for selecting patients for treatment with MET inhibitors. MET, the HGF receptor, is encoded by the MET proto-oncogene that may be activated by mutation or amplification. MET expression and activity were tested in a panel of control cell lines. MET could be detected in formalin fixed paraffin, embedded (FFPE) samples by IHC and RNA ISH, and this was confirmed by sandwich immunoassays of fresh frozen samples. Gastric cancer cell lines with high MET expression and phosphorylation of tyrosine-1349 respond to the MET inhibitor, BAY-853474. High expression and phosphorylation of MET is a predictive biomarker for response to MET inhibitors. We then analyzed MET expression and activity in a matched set of FFPE vs. fresh frozen tumor samples consisting of 20 cases of gastric cancer. Two of 20 clinical samples investigated exhibited high MET expression with RNA ISH and IHC. Both cases were shown by sandwich immunoassays to exhibits strong functional activity. Expression levels and functional activity in these two cases were in a range that predicted response to treatment. Our findings indicate that owing to its high selectivity, RNA ISH can be used to confirm findings obtained by IHC and potentially may replace IHC for certain targets if no suitable antibodies are available. RNA ISH is a valid platform for testing predictive biomarkers for patient selection.     

Assessment of antioxidant activities in roots of Miswak (Salvadora persica) plants grown at two different locations in Saudi Arabia

Traditionally, in Middle Eastern countries, many cultures use chewing sticks of arak for medicinal purposes especially, for oral cleanliness care. It was used by Muslims for treatment of teeth and highly recommended to be used by Muslims during the whole day. Therefore, the present work aimed to determine the total phenolic content and total flavonoids in two Miswak extracts obtained from arak roots collected from two different localities in Saudi Arabia. They were extracted with aqueous ethanol (80%) and used to estimate in vitro their antioxidative abilities. The new findings showed that the two tested extracts contained significantly different amounts of both total phenolic content and total flavonoids. According to the increase of total phenolic contents and total flavonoids obtained from the two extracts, Miswak collected from the southern region was found to contain more contents than those collected from the middle region. The results of antioxidant activities of Miswak root extract obtained by using different in vitro methods were varied depending on the technique used. According to the malondialdehyde (MDA) method, hydrogen peroxide (H₂O₂) scavenging ability and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods, the two Miswak extracts exhibited to have high to very high antioxidant activities. Mostly, the values of antioxidant activities of Southern region have been shown to be always the highest.     

Contrasting evolutionary rates in the duplicate chaperonin genes of Mycobacterium tuberculosis and M. leprae

A phylogenetic analysis of chaperonin (heat shock protein 60) sequences from prokaryotes and eukaryotes indicated that a single gene duplication event in the common ancestor of Mycobacterium tuberculosis, M. leprae, and Streptomyces albus gave rise to the duplicate chaperonin genes found in these species (designated HSP65 and GroEL in the mycobacterial species). Comparison of rates of synonymous and nonsynonymous nucleotide substitution in different gene regions suggested that the 5' end of the HSP65 gene was homogenized by an ancient recombination event between M. tuberculosis and M. leprae. In S. albus, the two duplicated chaperonin genes have evolved at essentially the same rate. In both M. tuberculosis and M. leprae, however, the GroEL gene has evolved considerably more rapidly at nonsynonymous nucleotide sites than has the HSP65 gene. Because this difference is not seen at synonymous sites, it must be due to a difference in selective constraint on the proteins encoded by the two genes, rather than to a difference in mutation rate. The difference between GroEL and HSP65 is striking in regions containing epitopes recognized by T cells of the vertebrate host; in certain cross-reactive epitopes conserved across all organisms, nonsynonymous sites in GroEL have evolved twice as fast as those in HSP65. It is suggested that these differences are correlated with differences in the way in which the duplicate chaperonins of M. tuberculosis and M. leprae interact with the host immune system.      

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.     

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.