High-performance liquid chromatographic assay of (±)-ethopropazine and its enantiomers in rat plasma

Two high-performance liquid chromatographic (HPLC) methods are described for determination of (±)-ethopropazine (ET) in rat plasma. After deproteination and liquid–liquid extraction, assay of (±)-ET was performed using either a C₁₈ column (non-stereospecific assay) or an (α-R-naphthyl)ethylurea column (stereospecific assay). The UV detection was at 250 nm. Mean recovery was >85%. Both assays demonstrated excellent linear relationships between peak height ratios and plasma concentrations; quantitation limits were ≤25 ng/ml, based on 100 μl rat plasma. Accuracy and precision were <17% with both methods. Both methods were applied successfully to the measurement of ET plasma concentrations in rats given the drug intravenously.     

Use of Nitroxides as NMR Contrast Enhancing Agents for Joints

NMR imaging is a well-established technology for obtaining cross-sectional anatomic pictures of organs and tissues. In addition, NMR can provide valuable information about the physiologic state of organs and tissues, especially, as a consequence of cellular injury. With this in mind, NMR in combination with gadolinium-based contrast enhancing agents has been used to assist in the detection of abnormalities to joints as well as to evaluate the status of damage resulting from an injury to this site. We describe the synthesis of a new nitroxide, which is bioresistant to the one-electron reduction mediated by superoxide in the presence of cysteine. This model mimics the reduction of nitroxides by extracellular secretion of superoxide by PMA-stimulated neutrophils. With this nitroxide, we found, in the range from 15 to 17.5μmoles, enhancement of an NMR image in the knee joint of rabbits. Of interest is the finding that the contrast image remained for at least 90 minutes. These results demonstrate the utility of nitroxides as contrast enhancing agents for NMR imaging of joints.     

Coordinated stimulation by triiodothyronine of fatty acid synthesis and isoproterenol-sensitive fatty acid release in two preadipocyte cell lines of lean or genetically obese mice

The development and thyroid hormone sensitivity of fatty acid and triacylglycerol synthesis from 14C-acetate and of the isoproterenol-sensitive fatty acid release, were studied in two preadipocyte cell lines during the adipose differentiation: the Ob 17 and the HGFu cell lines cloned from the periepididymal adipose tissue of adult mice genetically obese and phenotypically lean respectively. Both parameters increased and peaked in the same time-period during the second week of culture after confluence. Both parameters were also amplified when T3 was added to the culture medium at confluence. The increment due to T3 was concentration dependent: it peaked at the physiological concentration of 1.5 nM and declined thereafter with the same pattern. This shows that some steps of two opposite pathways of lipid metabolism in differentiating preadipose cells can be stimulated by triiodothyronine in a similar manner and suggests a coordinated regulation. No significant difference could be detected between cells from lean or genetically obese mice.     

Methods for the identification of N-asparaginyl and O-seryl/threonyl glycosidic linkages to aminosugars in glycoproteins

Methods are presented for the identification of certain glycopeptide bonds in glycoproteins. Mucin-type linkages are determined following treatment of glycoproteins with alkaline sodium [³H]borohydride. Such treatment cleaves O-glycosidic bonds to serine and threonine and simultaneously labels the sugar and amino acid components of the linkage. Following acid hydrolysis and dansylation, the sugar component of the linkage is identified as its corresponding dansyl-hexosaminitol by fluorographic techniques. A method is described for the separation of dansyl-galactosaminitol and dansyl-glucosaminitol by thin-layer electrophoresis in borate buffers. The amino acid component of the glycopeptide linkage is identified by fluorography following two-dimensional thin-layer chromatography of its dansyl derivative on polyamide plates. For the analysis of plasma-type glycoproteins, glycopeptides are prepared by exhaustive pronase digestion and purified by gel filtration chromatography. Final purification is effected by dansylation and thin-layer electrophoresis. The linkage compound 2-acetamido-1-N-β-l-aspartyl-2-deoxy-β-d-glucopyranosylamine is isolated from such glycopeptides as its dansyl derivative following partial acid hydrolysis. Its identity is confirmed by comparison of its properties with those of the synthetic compound. Thus the components of the glycosylamine linkage are identified following complete acid hydrolysis, redansylation, and separation by thin-layer electrophoresis.     

Natural Fiber Welded Electrode Yarns for Knittable Textile Supercapacitors

Natural fiber welded (NFW) yarns embedded with porous carbon ­materials are described for applications as electrodes in textile electrochemical capacitors. With this fabrication technique, many kinds of carbons can be embedded into cellulose based yarns and subsequently knitted into full ­fabrics on industrial knitting machines. Yarns welded with carbon and ­stainless steel have device capacitances as high as 37 mF cm^‐1, one of the highest reported values for carbon‐based yarns. The versatility of this ­technique to weld any commercially available cellulose yarn with any ­micro‐ or nanocarbon means properties can be tuned for specific applications. Most importantly, it is found that despite having full flexibility, increased strength, and good electrochemical performance, not all of the electrode yarns are ­suitable for knitting. Therefore, it is recommended that all works reporting on fiber/yarn capacitors for wearables attempt processing into full fabrics.     

Combination of two chromophores: synthesis and PDT application of porphyrin-pentamethinium conjugate

A general method for the synthesis of a novel porphyrin with pentamethine periphery substitution is described. The combination of two chromophoric systems, a porphyrin macrocycle and a polymethine moiety was achieved by transformation of tetrapyridyl porphyrin. The synthetic strategy included conversion of the tetrapyridyl porphyrin to its corresponding 2,4-dinitrophenylpyridinuim salt, which was subsequently converted to tetrakis(meso-pentamethinium salt) on the porphyrin core. This novel porphyrin exhibited PDT properties as manifested by the induction of apoptosis in the myeloid cell line HL-60 and the effective reduction of amelanotic melanoma in nude mice.     

Screening of enzymatic activities for the depolymerisation of the marine bacterial exopolysaccharide HE800

The exopolysaccharide (EPS) HE800 is a marine-derived polysaccharide (from 8 × 10(5) to 1.5 × 10(6) g mol(-1)) produced by Vibrio diabolicus and displaying original structural features close to those of glycosaminoglycans. In order to confer new biological activities to the EPS HE800 or to improve them, structural modifications need to be performed. In particular, depolymerisation is required to generate low-molecular-weight derivatives. To circumvent the use of chemical methods that lack specificity and reproducibility, enzymes able to perform such reaction are sought. This study reports the screening for enzymes capable of depolymerising the EPS HE800. A large diversity of enzyme sources has been studied: commercially available glycoside hydrolases with broad substrate specificity, lyases, and proteases as well as growing microorganisms. Interestingly, we found that the genus Enterococcus and, more particularly, the strain Enterococcus faecalis were able to depolymerise the EPS HE800. Partial characterization of the enzymatic activity gives evidence for a random and incomplete depolymerisation pattern that yields low-molecular-weight products of 40,000 g mol(-1). Genomic analysis and activity assays allowed the identification of a relevant open reading frame (ORF) which encodes an endo-N-acetyl-galactosaminidase. This study establishes the foundation for the development of an enzymatic depolymerisation process.     

The bacterial Tn<Emphasis Type="Italic">9</Emphasis> chloramphenicol resistance gene: an attractive DNA segment for <Emphasis Type="Italic">Mos1 mariner</Emphasis> insertions

The eukaryotic mariner transposons are currently thought to have no sequence specificity for integration other than to insert within a TA contained in a degenerated [TA]_1–4 tract, either in vitro or in vivo. We have investigated the properties of a suspected hotspot for the integration of the mariner Mos1 element, namely the Tn9 cat gene that encodes a chloramphenicol acetyl transferase. Using in vitro and bacterial transposition assays, we confirmed that the cat gene is a preferential target for MOS1 integration, whatever its sequence environment, copy number or chromosomal locus. We also observed that its presence increases transposition rates both in vitro and in bacterial assays. The structural and sequence features that constitute the attractiveness of cat were also investigated. We first demonstrated that supercoiling is essential for the cat gene to be a hot spot. In contrast to the situation for Tc1-like elements, DNA curvature and bendability were not found to affect integration target preferences. We found that Mos1 integrations do not occur randomly along the cat gene. All TA dinucleotides that are preferred for integration were found within either TATA or TA×TA motifs. However, these motifs are not sufficient to constitute an attractive dinucleotide, since four TATA and TA×TA sites are cold spots. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of Pulsatile Retinal Movements by Spectral-Domain Low-Coherence Interferometry: Influence of Age and Glaucoma on the Pulse Wave

Recent studies have shown that ocular hemodynamics and eye tissue biomechanical properties play an important role in the pathophysiology of glaucoma. Nevertheless, better, non-invasive methods to assess these characteristics in vivo are essential for a thorough understanding of degenerative mechanisms. Here, we propose to measure ocular tissue movements induced by cardiac pulsations and study the ocular pulse waveform as an indicator of tissue compliance. Using a novel, low-cost and non-invasive device based on spectral-domain low coherence interferometry (SD-LCI), we demonstrate the potential of this technique to differentiate ocular hemodynamic and biomechanical properties. We measured the axial movement of the retina driven by the pulsatile ocular blood flow in 11 young healthy individuals, 12 older healthy individuals and 15 older treated glaucoma patients using our custom-made SD-OCT apparatus. The cardiac pulse was simultaneously measured through the use of an oximeter to allow comparison. Spectral components up to the second harmonic were obtained and analyzed. For the different cohorts, we computed a few parameters that characterize the three groups of individuals by analyzing the movement of the retinal tissue at two locations, using this simple, low-cost interferometric device. Our pilot study indicates that spectral analysis of the fundus pulsation has potential for the study of ocular biomechanical and vascular properties, as well as for the study of ocular disease.     

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy,Viral Evolution and Compartmentalization

Background

Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain.

Methods & Results

We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.

Conclusions

The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.