A Synthesis of New Rigid Fluorescent Bichromophoric Probes for Studying Mechanisms of Donor-Donor Energy Migration

Three new fluorescent probes were synthesized for improving the method of studying donor-donor energy migration (DDEM). Each probe has two identical fluorescent 7-diethylaminocoumarin-3-carbonyl groups attached to a rigid bisteroid dodecacyclic spacer through additional inserts. In two probes, the inserts are β-Ala and L-Ser residues, which provide for a different nearest environment of the fluorophores. The third probe has identical β-Ala inserts.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 331–334.Original Russian Text Copyright © 2005 by Boldyrev, Molotkovsky.     

19F NMR measurements of NO production in hypertensive ISIAH and OXYS rats

Recently we demonstrated the principal possibility of application of 19F NMR spin-trapping technique for in vivo *NO detection [Free Radic. Biol. Med. 36 (2004) 248]. In the present study, we employed this method to elucidate the significance of *NO availability in animal models of hypertension. In vivo *NO-induced conversion of the hydroxylamine of the fluorinated nitronyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) in hypertensive ISIAH and OXYS rat strains and normotensive Wistar rat strain was measured. Significantly lower HIN/HNN ratios were measured in the blood of the hypertensive rats. The NMR data were found to positively correlate with the levels of nitrite/nitrate evaluated by Griess method and negatively correlate with the blood pressure. In comparison with other traditionally used methods 19F NMR spectroscopy allows in vivo evaluation of *NO production and provides the basis for in vivo *NO imaging.     

In vivo detection of secreted proteins from wounded skin using capillary ultrafiltration probes and mass spectrometric proteomics

The identification of in vivo secreted proteins is a major challenge in systems biology. Here we report a novel technique using capillary ultrafiltration (CUF) probes to identify the secreted proteins involved in wound healing. CUF probes, which use semipermeable membrane hollow fibers to continuously capture secreted proteins, were used to sample skin wound fluids. To identify low-abundance proteins, we digested the CUF probe-collected wound fluid with trypsin and then directly subjected it to MS without using 2-DE separation. Two protein fragments with masses of 1565.7 and 1694.8 Da were identified by MS as peptides of thymosin beta10 and beta4, respectively. This is the first identification of thymosin beta10 as an in vivo constituent of the skin wound fluid. The LKKTETQ peptide, a common actin-binding domain of thymosin beta4 and beta10, significantly enhanced skin wound healing in vitro and in vivo. Our data suggest that the enhancement of wound healing by LKKTETQ may be mediated by purinergic receptors. The technique of using CUF probes linked to mass spectrometric proteomics represents a powerful method to identify in vivo secreted proteins, and may be applicable for identification of proteins relevant in various human diseases.     

Interaction of delta sleep-inducing peptide and its analogues with cellular membranes: A structure-function analysis

The possibility of a correlation between the membrane properties of the delta sleep-inducing peptide (DSIP) and its analogues and their biological activity in vivo was examined by a comparative study of the membrane effects of these peptides. The peptides exhibiting biological activity in vivo were shown to cause a statistically reliable disordering of lipids in thrombocyte plasma membranes similar to the effect of DSIP. The membrane effect of the D-Val²-, D-Tyr²-, and Tyr¹, Pro² analogues of DSIP had the same bimodal dose dependence characteristic of natural DSIP. Only a slight nonspecific lipid disordering was registered for Trp-Asp-Ala-Ser-Gly-Glu, a biologically inactive hexapeptide analogue. These results indicate a correlation between the biological activity of the peptides during in vivo tests and their membrane properties in vitro. The structure-function relationship was studied within the group of DSIP analogues examined in vitro. The DSIP modeling effect, especially pronounced under the action of stress factors, was suggested to be directly associated with the ability of DSIP to change the dynamic structure of biological membranes.     

A molecular revision of the taxonomic status of mermithid parasites of black flies from Quebec (Canada)

The four currently recognized mermithid (Nematoda) species parasitizing black flies (Diptera: Simuliidae) from Northeast America were distinguished using discriminatory PCR primers aimed at COI and 18S rDNA. Isomermis wisconsinensis, Gastromermis viridis and Mesomermis camdenensis were easily differentiated using either genomic target, even for juvenile mermithids damaged beyond morphological recognition. However, specimens from Mesomermis flumenalis being identical in external morphology and producing a unique-sized PCR product were classified by sequence data into four clearly distinguished molecular variants. This quartet was made of two winter and two summer ‘physiological variants’, including one which also belonged to, but diverged early from the rest of the Mesomermis genus. Combining the multiplex PCR and sequencing approaches allowed for the characterization of a multiple parasitism which simultaneously implicated I. wisconsinensis and two M. flumenalis variants. With another instance where parasites were identified by morphology only, this is the first report of black fly parasitism by multiple mermithid species. A phylogenetic tree built by combining our sequences to previous GenBank entries likely indicates a monophyletic origin for the mermithid family, but also suggests that differentiation between parasite genera sometimes occurred before the evolutionary emergence of the actual host group.     

phylochipanalyser - a program for analysing hierarchical probe sets

The recent introduction of phylochips that contain molecular probes facilitates environmental microbial identification in a single experiment without previous cultivation. A set of probes recognizing species at different taxonomic levels is denoted as a hierarchical set. Application of hierarchical probe sets on a DNA microarray allows the assessment of biodiversity with different resolutions. It significantly increases the robustness of the results retrieved from phylochip experiments because of the possible consistency checks of hybridization across different taxonomic levels. Here, we present a computer program, phylo-chipanalyser, for the hierarchy editing and the evaluation of phylochip data generated from hierarchical probe sets.     

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.     

Calcium and pH patterning at the apical meristem are specifically altered by photoperiodic flower induction in Chenopodium spp.

The apical meristem of the short‐day plant Chenopodium rubrum responds to photoperiodic flower induction with specific changes of pH and Ca²⁺ patterning immediately after the inductive dark span. The red–far‐red reversibility of the pH and Ca²⁺ patterning in response to night break treatments was measured in order to distinguish between the effect of the prolonged dark span per se and the specific effect of photoperiodic flower induction. In addition, the pH and Ca²⁺ patterning in C. rubrum was compared with the long‐day plant Chenopodium murale. The pH was visualized using the fluorescent probe carboxy SNARF‐1. Calcium ion concentrations were studied using a combination of Ca²⁺‐probes Fluo‐3 and Fura Red. It was observed that the specific changes in pH and Ca²⁺ patterning at the apical meristem of C. rubrum were abolished by the red‐light break. This effect was fully reversed with a subsequent single far‐red treatment. These observations infer the influence of phytochrome on both pH and Ca²⁺ patterning. Changes in pH and Ca²⁺ patterning upon flower induction were observed in both long‐day and short‐day plants. These results support the hypothesis that changes of pH and [Ca²⁺] in cells of the apical meristem are part of the pathway in signal transduction triggering flower initiation.     

Effectiveness of TaqMan probes for detection of fish eggs and larvae in the stomach contents of a teleost predator

Experiments were conducted on the ability of TaqMan molecular probes to detect plaice Pleuronectes platessa DNA from eggs, and cod Gadus morhua DNA from eggs and larvae following ingestion by a teleost predator, whiting Merlangius merlangus. Estimated half-life detection rate (T50) for eggs was 31 h, and 26 h for larvae, with some positive detections occurring even after visual inspection indicated complete gut clearance. Because TaqMan probes are taxon specific, the results presented demonstrate that this technique can provide a means of rapid and unambiguous detection of predation by teleosts on fish eggs and larvae.     

A sensitive,specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivax and Plasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivax and Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed with Plasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochrome b-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.