CEP Biomarkers as Potential Tools for Monitoring Therapeutics

Background

Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids that are elevated in ocular tissues and plasma in age-related macular degeneration (AMD) and in rodents exposed to intense light. The goal of this study was to determine whether light-induced CEP adducts and autoantibodies are modulated by pretreatment with AL-8309A under conditions that prevent photo-oxidative damage of rat retina. AL-8309A is a serotonin 5-HT_1A receptor agonist.

Methods

Albino rats were dark adapted prior to blue light exposure. Control rats were maintained in normal cyclic light. Rats were injected subcutaneously 3x with 10 mg/kg AL-8309A (2 days, 1 day and 0 hours) before light exposure for 6 h (3.1 mW/cm², λ=450 nm). Animals were sacrificed immediately following light exposure and eyes, retinas and plasma were collected. CEP adducts and autoantibodies were quantified by Western analysis or ELISA.

Results

ANOVA supported significant differences in mean amounts of CEP adducts and autoantibodies among the light + vehicle, light + drug and dark control groups from both retina and plasma. Light-induced CEP adducts in retina were reduced ~20% following pretreatment with AL-8309A (n = 62 rats, p = 0.006) and retinal CEP immunoreactivity was less intense by immunohistochemistry. Plasma levels of light-induced CEP adducts were reduced at least 30% (n = 15 rats, p = 0.004) by drug pretreatment. Following drug treatment, average CEP autoantibody titer in light exposed rats (n = 22) was unchanged from dark control levels, and ~20% (p = 0.046) lower than in vehicle-treated rats.

Conclusions

Light-induced CEP adducts in rat retina and plasma were significantly decreased by pretreatment with AL-8309A. These results are consistent with and extend previous studies showing AL-8309A reduces light-induced retinal lesions in rats and support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics.     

Copper(II) complexes with chicken prion repeats: influence of proline and tyrosine residues on the coordination features

The prion protein (PrP^c) is a copper-binding glycoprotein that can misfold into a β-sheet-rich and pathogenic isoform (PrP^sc) leading to prion diseases. The first non-mammalian PrP^c was identified in chicken and it was found to keep many structural motifs present in mammalian PrP^c, despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY , HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV–vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in June 2005 with corrections to the text. The author name LaMendola has been corrected to La Mendola.     

Rapid and sensitive NMR method for osmolyte determination

We propose a rapid and sensitive method for osmolyte determination, based on one-dimensional and two-dimensional 1H NMR spectroscopy applied directly on culture of haloalkalophilic Halomonas pantelleriensis and acidothermophilic archaeon Sulfolobus solfataricus, without any extraction procedure. The osmoprotectants hydroxyectoine, ectoine, glutamate, glycine-betaine and treahalose can easily be quantified by integrating the peak areas with respect to an internal standard, and the concentrations evaluated with this method are in excellent agreement with the values previously reported. Furthermore, trace amount of osmoprotectants, often undetectable after extraction procedures, can also be evaluated.     

Ultrastructural analysis of the aberrant axoneme morphogenesis in thrips (Thysanoptera, Insecta)

Thrips spermiogenesis is characterized by unusual features in the differentiating spermatid cells. Three centrioles from which three individual short flagella are initially assembled, make the early spermatid a tri-flagellated cell. Successively, during spermatid maturation, the three basal bodies maintain a position close to the most anterior end of the elongating nucleus, so that the three axonemes are progressively incorporated in the spermatid cytoplasm, where they run in parallel to the main nuclear axis. Finally, the three axonemes amalgamate to form a microtubular bundle. The process starts with the formation of rifts at three specific points in each axonemal circumference, corresponding to sites 1,3,7 and leads to the formation of 9 microtubular rows of different length, i.e. 3 "dyads", 3 "triads" and 3 "tetrads". In the spermatozoon, the nucleus, the mitochondrion and the bundle of microtubules are arranged in a helicoidal pattern. The elongation of the spermatozoon is allowed by the deep anchorage of the spermatid to the cyst cell through a dense mass of material which, at the end of spermiogenesis, becomes a long anterior cylindrical structure. This bizarre "axoneme" does not show any trace of progressive movement but it is able to beat. According to the presence of dynein arms, sliding can take place only within each row and not between the rows. The possible molecular basis underlying the peculiar instability of thrips axonemes is discussed in light of the present knowledge on the organization of the axoneme in mutant organisms carrying alterations of the tubulin molecule.     

<Emphasis Type="Italic">In vitro</Emphasis> plantlet production of the endangered <Emphasis Type="Italic">Pinguicula vulgaris</Emphasis>

This study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free ¼MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.     

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.     

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.     

Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping

Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.