Losartan inhibits endothelial-to-mesenchymal transformation in mitral valve endothelial cells by blocking transforming growth factor-β-induced phosphorylation of ERK

Adult cardiac valve endothelial cells (VEC) undergo endothelial to mesenchymal transformation (EndMT) in response to transforming growth factor-β (TGFβ). EndMT has been proposed as a mechanism to replenish interstitial cells that reside within the leaflets and further, as an adaptive response that increases the size of mitral valve leaflets after myocardial infarction. To better understand valvular EndMT, we investigated TGFβ-induced signaling in mitral VEC, and carotid artery endothelial cells (CAEC) as a control. Expression of EndMT target genes α-smooth muscle actin (α-SMA), Snai1, Slug, and MMP-2 were used to monitor EndMT. We show that TGFβ-induced EndMT increases phosphorylation of ERK (p-ERK), and this is blocked by Losartan, an FDA-approved antagonist of the angiotensin II type 1 receptor (AT1), that is known to indirectly inhibit phosphorylation of ERK (p-ERK). Blocking TGF-β-induced p-ERK directly with the MEK1/2 inhibitor RDEA119 was sufficient to prevent EndMT. In mitral VECs, TGFβ had only modest effects on phosphorylation of the canonical TGF-β signaling mediator mothers against decapentaplegic homolog 3 (SMAD3). These results indicate a predominance of the non-canonical p-ERK pathway in TGFβ-mediated EndMT in mitral VECs. AT1 and angiotensin II type 2 (AT2) were detected in mitral VEC, and high concentrations of angiotensin II (AngII) stimulated EndMT, which was blocked by Losartan. The ability of Losartan or MEK1/2 inhibitors to block EndMT suggests these drugs may be useful in manipulating EndMT to prevent excessive growth and fibrosis that occurs in the leaflets after myocardial infarction.     

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 μg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 μg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.     

Using genetic and morphological analysis to distinguish endangered taxa from their hybrids with the cultivated exotic pest plant <Emphasis Type="Italic">Lantana strigocamara</Emphasis> (syn: <Emphasis Type="Italic">Lantana camara</Emphasis>)

Because highly invasive species can rapidly assimilate rare taxa, we questioned whether two Florida endangered Lantana depressa varieties existed 21 years after Sanders documented their widespread hybridization with exotic Lantana strigocamara, and whether morphological traits could accurately discriminate genetic individuals. Stepwise discriminant analysis of morphological characters discriminated the three taxa, correctly classifying 98, 91, 89% of L. strigocamara, L. depressa var. depressa, and var. floridana. Hybrids blurred taxonomic distinctions of varieties and reduced classification accuracy by 7–17%. Species-specific Random Fragment Length Polymorphism (RFLP-PCR) confirmed hybridization has occurred. Intersimple Sequence Repeat (ISSR) fingerprints analyzed with STRUCTURE identified three groups indicating introgression. Morphological traits significantly, but weakly correlated with q ratios (P = 0.0001; r ² = 0.45). Although L. strigocamara introgression is widespread and ongoing, wild populations contain individuals that are predominantly L. depressa genome, supporting actions to remove adventive L. strigocamara, prevent its sale, and promote sales of genetically confirmed natives.     

Major histocompatibility complex variation and evolution at a single, expressed DQA locus in two genera of elephants

Genes of the vertebrate major histocompatibility complex (MHC) are crucial to defense against infectious disease, provide an important measure of functional genetic diversity, and have been implicated in mate choice and kin recognition. As a result, MHC loci have been characterized for a number of vertebrate species, especially mammals; however, elephants are a notable exception. Our study is the first to characterize patterns of genetic diversity and natural selection in the elephant MHC. We did so using DNA sequences from a single, expressed DQA locus in elephants. We characterized six alleles in 30 African elephants (Loxodonta africana) and four alleles in three Asian elephants (Elephas maximus). In addition, for two of the African alleles and three of the Asian alleles, we characterized complete coding sequences (exons 1–5) and nearly complete non-coding sequences (introns 2–4) for the class II DQA loci. Compared to DQA in other wild mammals, we found moderate polymorphism and allelic diversity and similar patterns of selection; patterns of non-synonymous and synonymous substitutions were consistent with balancing selection acting on the peptides involved in antigen binding in the second exon. In addition, balancing selection has led to strong trans-species allelism that has maintained multiple allelic lineages across both genera of extant elephants for at least 6 million years. We discuss our results in the context of MHC diversity in other mammals and patterns of evolution in elephants.     

Development and validation of a FACS-based lipoprotein localization screen in the Lyme disease spirochete <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

Background  

In our previous studies on lipoprotein secretion in the Lyme disease spirochete Borrelia burgdorferi, we used monomeric red fluorescent protein 1 (mRFP1) fused to specifically mutated outer surface protein A (OspA) N-terminal lipopeptides to gather first insights into lipoprotein sorting determinants. OspA:mRFP1 fusions could be detected by epifluorescence microscopy both in the periplasm and on the bacterial surface. To build on these findings and to complement the prior targeted mutagenesis approach, we set out to develop a screen to probe a random mutagenesis expression library for mutants expressing differentially localized lipoproteins.     

Identification of genes involved in the mutualistic colonization of the nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> by the bacterium <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>

Background  

Photorhabdus are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family Heterorhabditis. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in Photorhabdus that are important for IJ colonization.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.     

Optimization of the Water-Insoluble Procedures for <Emphasis Type="Italic">USP</Emphasis> General Chapter <Emphasis Type="Italic">Residual Solvents</Emphasis> <467>

The water-insoluble procedures in US Pharmacopeia (USP) General Chapter Residual Solvents <467>, which are based on European Pharmacopoeia procedures, were optimized and modified before their inclusion in the chapter to improve their scope, performance, and ruggedness. The optimized procedures use a static headspace introduction system with a gas chromatograph equipped with a flame ionization detector. This article describes some of the key changes made to the USP published procedures, including use of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) as the solvent, addition of 5 mL of water and 1 mL of sample (dissolved in DMSO or DMF) to the headspace vial, use of a 3:1 GC split ratio, and use of new matrix-matched system suitability solutions. These procedures were verified with two different active pharmaceutical ingredients—hydroxyzine pamoate and prednisone. In the investigation, the more polar material (hydroxyzine pamoate) showed greater recoveries for the optimized procedures when prepared in DMSO. The less polar material (prednisone) typically had greater recoveries in DMF for the optimized procedures. During experimentation, insights into sample preparation, additional types of headspace instrumentation, solvent purity, and other parameters were also gained.     

Sex-determined differential mortality of milkweed bugs, Oncopeltus fasciatus (hemiptera), to aflatoxins

Studies on the aflatoxins, toxic metabolites of Aspergillus flavus and A. parasiticus, have involved test systems ranging from cell cultures to laboratory animals. This work reports on the differential response by sex of Oncopeltus fasciatus to aflatoxin B₁ (AFB₁). Young adult milkweed bugs were chosen randomly from our stock colony and housed in glass culture jars. Triplicate sets of experimental animals were fed 5 μg/ml of AFB₁ in their liquid diet. The first death for the experimental females occurred at day 4, and at 10 days for the experimental males. A 50% lethality level for experimental females developed by day 8. Males subjected to the same concentration achieved a 50% lethality level at day 24. For the females the LD₅₀ occurred after consuming 0.49 μg/ml of AFB₁. The results indicate that adult female milkweed bugs were hypersensitive to AFB₁ as compared to adult males. This organism is more sensitive than the American cockroach and less sensitive than the fruitfly, housefly, and honeybee to toxic aflatoxicosis. Even the female is not sufficiently sensitive to rate highly as a bioassay organism for AFB₁. The extreme difference in mortality between the sexes is significant, unusual, and unexplained.     

p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.