A Reaction-Based Optical Fingerprinting Strategy for the Recognition of Fat-Soluble Samples: Discrimination of Motor Oils.

Optical "fingerprints" are widely used for chemometrics-assisted recognition of samples of different types. An emerging trend in this area is the transition from obtaining "static" spectral data to reactions analyzed over time. Indicator reactions are usually carried out in aqueous solutions; in this study, we developed reactions that proceed in an organic solvent, thereby making it possible to recognize fat-soluble samples. In this capacity, we used 5W40, 10W40, and 5W30 motor oils from four manufacturers, with six samples in total. The procedure involved mixing a dye, sample, and reagents (HNO3, HCl, or tert-butyl hydroperoxide) in an ethanolic solution in a 96-well plate and measuring absorbance or near-infrared fluorescence intensity every several minutes for 20-55 min. The obtained photographic images were processed by linear discriminant analysis (LDA) and the k-nearest neighbors algorithm (kNN). Discrimination accuracy was evaluated by a validation procedure. A reaction of oxidation of a dye by nitric acid allowed us to recognize all six samples with 100% accuracy for LDA. Merging of data from the four reactions that did not provide complete discrimination ensured an accuracy of 93% for kNN. The newly developed indicator systems have good prospects for the discrimination of other fat-soluble samples. Overall, the results confirm the viability of the kinetics-based discrimination strategy.

Carbocyanine-Based Optical Sensor Array for the Discrimination of Proteins and Rennet Samples Using Hypochlorite Oxidation.

Optical sensor arrays are widely used in obtaining fingerprints of samples, allowing for solutions of recognition and identification problems. An approach to extending the functionality of the sensor arrays is using a kinetic factor by conducting indicator reactions that proceed at measurable rates. In this study, we propose a method for the discrimination of proteins based on their oxidation by sodium hypochlorite with the formation of the products, which, in turn, feature oxidation properties. As reducing agents to visualize these products, carbocyanine dyes IR-783 and Cy5.5-COOH are added to the reaction mixture at pH 5.3, and different spectral characteristics are registered every several minutes (absorbance in the visible region and fluorescence under excitation by UV (254 and 365 nm) and red light). The intensities of the photographic images of the 96-well plate are processed by principal component analysis (PCA) and linear discriminant analysis (LDA). Six model proteins (bovine and human serum albumins, γ-globulin, lysozyme, pepsin, and proteinase K) and 10 rennet samples (mixtures of chymosin and pepsin from different manufacturers) are recognized by the proposed method. The method is rapid and simple and uses only commercially available reagents.

Hemostasis System and Plasminogen Activity in Retrochorial Hematoma in the First Trimester of Pregnancy.

(1) Background: The components of the fibrinolytic system and its main component, plasminogen, play a key role in the first months of pregnancy. The effect of autoantibodies interacting with plasminogen in the formation of retrochorial hematoma is unknown. The aim of our study was to determine the role of plasminogen and IgA, IgM, and IgG, which bind to plasminogen, in retrochorial hematoma. (2) Methods: Prothrombin time (PT), thrombin time (TT), partial activated thromboplastin time (aPTT), soluble fibrin-monomer complex (SFMC), D-dimer, plasminogen activity (%Plg), plasminogen concentration (Plg), and the levels of IgG (IgG-Plg), IgM (IgM-Plg), IgA (IgA-Plg) interacting with plasminogen were determined in plasma samples of 57 women with normal pregnancy and 16 with retrochorial hematoma. (3) Results: %Plg in plasma samples from women with retrochorial hematoma was significantly lower than in plasma samples from women with normal pregnancy. The diagnostic significance of %Plg in the ROC analysis was AUC = 0.85. A direct correlation was found between aPTT and the level of autologous IgM interacting with plasminogen. (4) Conclusions: A decrease in the activity of plasminogen in the blood serum of women in the first trimester of pregnancy may indicate disturbances in the hemostasis system and the formation of retrochorial hematoma. According to the results of the study, it is possible to recommend the determination of plasminogen activity in the management of pregnant women in gynecological practice.

Proteolyzed Variant of IgG with Free C-Terminal Lysine as a Biomarker of Prostate Cancer.

The differential diagnosis of prostate cancer is problematic due to the lack of markers with high diagnostic accuracy. We previously demonstrated the increased binding of IgG to human plasminogen (PLG) in plasma of patients with prostate cancer (PC) compared to healthy controls. Heavy and light chains of PLG (PLG-H and PLG-L) were immobilized on 96-well plates and the binding of IgG to PLG-H and PLG-L was analyzed in serum from 30 prostate cancer (PC) patients, 30 patients with benign prostatic hyperplasia (BPH) and 30 healthy controls using enzyme-linked immunosorbent assay (ELISA). Our results demonstrate that IgG from PC sera bind to PLG-H but not to PLG-L. This interaction occurred through the free IgG C-terminal lysine (Lys) that becomes exposed as a result of IgG conformational changes associated with proteolysis. Circulating levels of modified IgG with exposed C-terminal Lys (IgG-Lys) were significantly higher in PC patients than in healthy controls and in BPH. We used Receiver Operating Characteristic (ROC) analysis to calculate the sensitivity (SN) and specificity (SP) of circulating IgG-Lys for differentiating PC from BPH as 77% and 90%, respectively. The area under the curve (AUC) was 0.87. We demonstrated that the diagnostic accuracy of circulating levels of IgG-Lys is much higher than diagnostic accuracy of total PSA (tPSA).

The exon junction complex factor Y14 is dynamic in the nucleus of the beetle Tribolium castaneum during late oogenesis.

BACKGROUND: The oocyte chromosomes of the red flour beetle, Tribolium castaneum, are gathered into a knot, forming a karyosphere at the diplotene stage of meiotic prophase. Chromatin rearrangement, which is a characteristic feature of oocyte maturation, is well documented. The T. castaneum karyosphere is surrounded by a complex extrachromosomal structure termed the karyosphere capsule. The capsule contains the vast majority of oocyte RNA. We have previously shown using a BrUTP assay that oocyte chromosomes in T. castaneum maintain residual transcription up to the very end of oocyte maturation. Karyosphere transcription requires evidently not only transcription factors but also mRNA processing factors, including the components of the exon junction complex with its core component, the splicing factor Y14. We employed a gene engineering approach with injection of mRNA derived from the Myc-tagged Y14 plasmid-based construct in order to monitor the newly synthesized fusion protein in the oocyte nuclei. RESULTS: Our preliminary data have been presented as a brief correspondence elsewhere. Here, we provide a full-length article including immunoelectron-microscopy localization data on Y14-Myc distribution in the nucleus of previtellogenic and vitellogenic oocytes. The injections of the fusion protein Y14-Myc mRNA into the oocytes showed a dynamic pattern of the protein distribution. At the previtellogenic stage, there are two main locations for the protein: SC35 domains (the analogues of interchromatin granule clusters or nuclear speckles) and the karyosphere capsule. At the vitellogenic stage, SC35 domains were devoid of labels, and Y14-Myc was found in the perichromatin region of the karyosphere, presumably at the places of residual transcription. We show that karyosphere formation is accompanied by the movement of a nuclear protein while the residual transcription occurs during genome inactivation. CONCLUSIONS: Our data indicate that the karyosphere capsule, being a destination site for a protein involved in mRNA splicing and export, is not only a specializes part of nuclear matrix separating the karyosphere from the products of chromosome activity, as believed previously, but represents a special nuclear compartment involved in the processes of gene expression in the case the karyosphere retains residual transcription activity.

Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality.

We performed immunofluorescent analysis of DNA hydroxymethylation and methylation in human testicular spermatogenic cells from azoospermic patients and ejaculated spermatozoa from sperm donors and patients from infertile couples. In contrast to methylation which was present throughout spermatogenesis, hydroxymethylation was either high or almost undetectable in both spermatogenic cells and ejaculated spermatozoa. On testicular cytogenetic preparations, 5-hydroxymethylcytosine was undetectable in mitotic and meiotic chromosomes, and was present exclusively in interphase spermatogonia Ad and in a minor spermatid population. The proportions of hydroxymethylated and non-hydroxymethylated diploid and haploid nuclei were similar among samples, suggesting that the observed alterations of 5-hydroxymethylcytosine patterns in differentiating spermatogenic cells are programmed. In ejaculates, a few spermatozoa had high 5-hydroxymethylcytosine level, while in the other ones hydroxymethylation was almost undetectable. The percentage of highly hydroxymethylated (5-hydroxymethylcytosine-positive) spermatozoa varied strongly among individuals. In patients from infertile couples, it was higher than in sperm donors (P<0.0001) and varied in a wider range: 0.12-21.24% versus 0.02-0.46%. The percentage of highly hydroxymethylated spermatozoa correlated strongly negatively with the indicators of good semen quality - normal morphology (r=-0.567, P<0.0001) and normal head morphology (r=-0.609, P<0.0001) - and strongly positively with the indicator of poor semen quality: sperm DNA fragmentation (r=0.46, P=0.001). Thus, the immunocytochemically detected increase of 5hmC in individual spermatozoa is associated with infertility in a couple and with deterioration of sperm parameters. We hypothesize that this increase is not programmed, but represents an induced abnormality and, therefore, it can be potentially used as a novel indicator of semen quality.

Design of molecular beacons: 3' couple quenchers improve fluorogenic properties of a probe in real-time PCR assay.

Convenient preparation of fluorogenic hairpin DNA probes (molecular beacons) carrying a pair of FAM fluorophores (located close to 5'-terminus of the probe) or a pair of BHQ1 quenchers on 3'-terminus (with (BHQ1)2 or BHQ1-BHQ1 composition) is reported. These probes were used for the first time in a real-time PCR assay and showed considerable improvements in fluorogenic properties (the total fluorescence increase or signal-to-background ratio) in assay conditions vs. conventional one-FAM-one-BHQ1 molecular beacon probes as well as vs. hydrolyzable one-FAM-one-BHQ1 TaqMan probes. At the same time, such multiple modifications of the probe do not influence its Cq (a fractional PCR cycle used for quantification). The probe MB14 containing a BHQ1-BHQ1 pair showed a PCR fluorescence/background value of 9.6 which is more than two times higher than that of a regular probe MB2 (4.6). This study demonstrates prospects for the design of highly fluorogenic molecular beacon probes suitable for quantitative real-time PCR and for other potential applications (e.g. intracellular RNA detection and SNP/mutation analysis).

Nuclear structures in Tribolium castaneum oocytes.

The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis.

Non-nucleoside phosphoramidites of xanthene dyes (FAM, JOE, and TAMRA) for oligonucleotide labeling.

This unit describes the preparation of 5- and 6-carboxy derivatives of the xanthene fluorescent dyes fluorescein (FAM), 4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE), and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion to non-nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. The use of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hydroxyls facilitates the chromatographic separation of isomers of carboxy-FAM and carboxy-JOE as pentafluorophenyl esters. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride gave a mixture of 4-dimethylamino-2-hydroxy-2',4'(5')-dicarboxybenzophenones, easily separable into individual compounds upon fractional crystallization. Individual isomeric benzophenones are precursors of 5- or 6-carboxytetramethylrhodamines. The dyes were converted into 6-aminohexanol- (JOE), 4-trans-aminocyclohexanol- (FAM and JOE), and hydroxyprolinol-based (TAMRA) phosphoramidite reagents.

4',5'-Dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE): synthesis and spectral properties of oligonucleotide conjugates.

A convenient procedure for the preparation of the fluorescent dye 4',5'-dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein (JOE) is reported; the overall yield achieved starting from isovanillin is 10 times higher (40% vs 4%) compared to the known procedure. Isomers (5- and 6-) are easily chromatographically separable as pentafluorophenyl esters of 3',6'-O-bis(cyclohexylcarbonyl) derivatives. Four non-nucleoside JOE phosphoramidites based on 5- and 6-isomers and flexible 6-aminohexanol (AH) or rigid 4-trans-aminocyclohexanol (ACH) linkers have been prepared and used for oligonucleotide labeling. Spectral and photophysical properties of 5'-JOE-modified oligonucleotides have been studied. Fluorescence quantum yield of the dye correlates with the nature of the linker (rigid vs flexible) and with the presence of dG nucleosides in close proximity to a JOE residue.

Practical synthesis of isomerically pure 5- and 6-carboxytetramethylrhodamines, useful dyes for DNA probes.

Simple and scalable synthesis of 5- and 6-carboxytetramethylrhodamines (TAMRAs) is reported. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride afforded a mixture of 4-dimethylamino-2-hydroxy-2',4'(5')-dicarboxybenzophenones, which can be easily separated into individual compounds upon recrystallization from methanol and acetic acid. Individual benzophenones were reacted with 3-dimethylaminophenol to give 5- or 6-carboxytetramethylrhodamines. The dyes were converted into hydroxyprolinol-based phosphoramidite reagents suitable for oligonucleotide synthesis. 5- and 6-TAMRA isomers on oligonucleotides showed similar absorption and emission spectra. Fluorescence quantum yield of the dyes correlates with the presence of dG nucleosides in the adjacent region of oligonucleotide sequence. Several energy transfer primers containing on their 5'-termini (6-FAM)dT(n)(6-TAMRA) dye system (n = 0, 2, 4, 6, 8, 10, 12, 14) were prepared, and their spectral properties were studied.

Universal nuclear domains of somatic and germ cells: some lessons from oocyte interchromatin granule cluster and Cajal body structure and molecular composition.

It is now clear that two prominent nuclear domains, interchromatin granule clusters (IGCs) and Cajal bodies (CBs), contribute to the highly ordered organization of the extrachromosomal space of the cell nucleus. These functional domains represent structurally stable but highly dynamic nuclear organelles enriched in factors that are required for different nuclear activities, especially RNA biogenesis. IGCs are considered to be the main sites for storage, assembly, and/or recycling of the essential spliceosome components. CBs are involved in the biogenesis of several classes of small RNPs as well as the modification of newly assembled small nuclear RNA. We have summarized data on the molecular composition, structure, and functional roles of IGCs and CBs in the nuclei of mammalian somatic cells and oocytes of some animals with a special focus on insects. We have focused on similarities and differences between the IGCs and CBs of oocytes and the well-studied CBs and IGCs of cultured mammalian somatic cells. We have shown the heterogeneous character of oocyte IGCs and CBs, both in structure and molecular content. We have also demonstrated the unique capacity of oocytes to form close structural interactions between IGC and CB components. We proposed to consider these joint structures as integrated entities, sharing the features of both IGCs and CBs.

Reactive trityl derivatives: stabilised carbocation mass-tags for life sciences applications.

The rational design of novel triarylmethyl (trityl)-based mass tags (MT) for mass-spectrometric (MS) applications is described. We propose a "pK(R+) rule" to correlate the stability of trityl carbocations with their MS performance: trityls with higher pK(R+) values ionise and desorb better. Trityl blocks were synthesised that have high pK(R+) values and are stable in conditions of MS analysis; these MTs can be ionised by matrix as well as irradiation with a 337 nm nitrogen laser. (13)C-Labelled tags were prepared for MS quantitation applications. Moreover, the tags were equipped with a variety of functional groups allowing conjugation with different functionalities within (bio)molecules to enhance the MS characteristics of the latter. The MS behaviour of model polycationic trityl compounds with and without the matrix was studied to reveal that poly-trityl clusters are always singly charged under the (MA)LDI-TOF conditions. Several peptide-trityl conjugates were prepared and comparisons revealed a beneficial effect of trityl tags on the conjugate detection in MS. Trityl compounds containing para-methoxy- and dimethylamine groups, as well as a xanthene fragment, showed considerable enhancement in MS detection of model peptides; thus they are promising tools for proteomic applications. Dimethoxytrityl derivatives allow one to distinguish between Arg- and Lys-containing peptides. Maleimido trityl derivatives are suitable for the efficient derivatisation of thiol-containing peptides in pyridine.

1-Phenylethynylpyrene (1-PEPy) as refined excimer forming alternative to pyrene: case of DNA major groove excimer.

1-Phenylethynylpyrene fluorochrome was studied as meta- and para-derivatives of arabino-uridine-2'-carbamates in ss and dsDNA. 1-PEPy showed red-shifted emission and increased fluorescence quantum yield compared to pyrene. Although 1-PEPy has very short excited lifetime (<2.5 ns), it is able to form inter- and intrastrand excimers on DNA, probably resulting from spatial preorganization of two dye molecules.

5(6)-carboxyfluorescein revisited: new protecting group, separation of isomers, and their spectral properties on oligonucleotides.

Pentafluorophenyl esters of 5- and 6-carboxyfluorescein-3',6'-O-dipivalate can be easily separated in multigram quantities by column chromatography. The individual isomers were converted into stable phosphoramidites suitable for oligonucleotide synthesis. The use of the cyclohexylcarbonyl (Chc) protecting group instead of pivaloyl (Piv) facilitates the separation of isomers. The fluorescence spectra of 5- and 6-carboxyfluoresceins on oligonucleotides were compared.

Cajal bodies and interchromatin granule clusters in cricket oocytes: composition, dynamics and interactions.

The organization and molecular composition of complicated Cajal bodies (CBs) and interchromatin granule clusters (IGCs) in oocytes of the house cricket, Acheta domesticus, were studied using immunofluorescent/confocal and Immunogold labeling/electron microscopy. In A. domesticus oocytes, the CB consists of the fibrillar matrix and a central cavity containing a predominantly granular body with insertions of tightly packed fibrillar material. The latter structure was identified as an "internal" IGC, since it is enriched with the SC35 protein, a marker for IGCs. The IGCs located outside the CB were also identified. Microinjections of the fluorescein-tagged U7 snRNA into the ooplasm showed the targeting of the U7 to the matrix of the CB. Some other essential CB components (coilin, snRNPs, fibrillarin) were found to be colocalized in the matrix of the CB. Neither confocal nor Immunogold microscopy revealed significant amounts of RNA polymerase II (pol II) in the CB of A. domesticus oocytes. The splicing factor SC35 was detected in the matrix of the CB. In oocytes treated with DRB, the amount of IGCs in the nucleoplasm increased significantly, granular and fibrillar components of IGCs were segregated, and the fibrillar areas accumulated pol II. Additionally, IG-like granules were shown to display on the surface of the CB probably due to a shifting from the internal IGC. We believe that in A. domesticus oocytes, CBs are involved in nuclear distribution of splicing factors, but their role in pol II transport is less significant. We also suggest that the formation of complicated CBs is a result of interconnection between two different nuclear domains, CBs and IGCs.

Interchromatin granule clusters in vitellogenic oocytes of the flesh fly, Sarcophaga sp.

Insect oocyte nuclei contain different extrachromosomal nuclear bodies including Cajal bodies and interchromatin granule clusters (IGCs). In the present study, we describe IGC equivalents in the vitellogenic oocytes of the flesh fly, Sarcophaga sp. These structures were found to consist of 20-40-nm granules and also include the fibrillar areas of high and low electron density. Immunogold labeling electron microscopy revealed IGC marker protein SC35, Sm proteins, and trimethylguanosine cap of small nuclear (sn) RNAs in these bodies. Antibody against the non-phosphorylated RNA polymerase II selectively labeled the fibrillar areas of low electron density located inside the IGCs.

5-Arylethynyl-2'-deoxyuridines, compounds active against HSV-1.

Three new 5-arylethynyl-2'-deoxyuridines containing bulky aryls have been prepared and tested against HSV-1 in Vero cells. The introduction of a substituent in the phenyl group of an inactive compound, 5-phenylethynyl-2'-deoxyuridine, leads to the appearance of anti-HSV properties. The most active compounds are those containing a polycyclic aromatic hydrocarbon residue attached to the 5 position of 2'-deoxyuridine through a rigid triple bond.

Effects of intravenous apolipoprotein A-I/phosphatidylcholine discs on paraoxonase and platelet-activating factor acetylhydrolase in human plasma and tissue fluid.

We have previously shown that intravenous apolipoprotein (apo) A-I/phosphatidylcholine (apo A-I/PC) discs increase plasma high-density lipoprotein (HDL) concentration in humans. We have now studied the associated changes in two enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH) that are carried in whole or in part by HDLs, and are thought to influence atherogenesis by hydrolyzing oxidized phospholipids in lipoproteins. Apo A-I/PC discs (40 mg/kg over 4 h) were infused into eight healthy males. Although plasma apo A-I and HDL cholesterol increased on average by 178 and 158%, respectively, plasma total PON and total PAF-AH concentrations did not rise. By the end of the infusion, HDL-associated PAF-AH had increased by 0.56 +/- 0.14 microg/mL (mean +/- S.D., P < 0.01), and nonHDL-associated PAF-AH had decreased by 0.84 +/- 0.11 microg/mL (P < 0.05). These changes were accompanied by an increase in the HDL-associated PAF-AH/apo A-I ratio from 0.19 to 0.35 (P < 0.05), and by a decrease in the nonHDL-associated PAF-AH/apo B ratio from 2.1 to 1.4 (P < 0.05). No changes in PON or PAF-AH concentrations were detected in prenodal lymph (tissue fluid), collected continuously from the leg. Our results show that the total concentrations of PON and PAF-AH in plasma are uninfluenced by plasma HDL concentration. PAF-AH transfers readily between HDLs and LDLs in vivo, and its distribution between them is determined partly by their relative concentrations and partly by HDL composition.