Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells

Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both ascorbic acid and dehydroascorbic acid to 10 to 20 pmol. This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441 from The American Cancer Society.     

Acidic Cave-Wall Biofilms Located in the Frasassi Gorge,Italy

Acidic biofilms present on cave walls in the sulfidic region of the Frasassi Gorge, Italy, were investigated to determine their microbial composition and their potential role in cave formation and ecosystem functioning. All biofilm samples examined had pH values < 1.0. Scanning electron microscopy of the biofilms revealed the presence of various filaments and rods associated in large clusters with mineral crystals. Qualitative energy-dispersive x-ray analysis was used to determine that the crystals present on the cave walls, associated with the microbial biofilm, were composed of calcium and barium sulfate. Ribosomal RNA-based methods to determine the microbial composition of these biofilms revealed the presence of at least two strains of potential acidophilic, sulfur-oxidizing bacteria, belonging to the genera Thiobacillus and Sulfobacillus. An acid-producing strain of Thiobacillus sp. also was obtained in pure culture. Stable isotope ratio analysis of carbon and nitrogen showed that the wall biofilms are isotopically light, suggesting that in situ chemoautotrophic activity plays an important role in this subsurface ecosystem.     

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.     

Unexpected intracellular localization of the AMD-associated cystatin C variant

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.     

Synthesis, liposomal preparation, and in vitro toxicity of two novel dodecaborate cluster lipids for boron neutron capture therapy

A new class of lipids, containing the closo-dodecaborate cluster, has been synthesized. Two lipids, S-(N, N-(2-dimyristoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-14) and S-(N, N-(2-dipalmitoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-16) are described. Both of them have a double-tailed lipophilic part and a headgroup carrying two negative charges. Differential scanning calorimetry shows that B-6-14 and B-6-16 bilayers have main phase transition temperatures of 18.8 and 37.9 degrees C, respectively. Above the transition temperature of 18.8 degrees C, B-6-14 can form liposomal vesicles, representing the first boron-containing lipid with this capability. Upon cooling below the transition temperature, stiff bilayers are formed. When incorporated into liposomal formulations with equimolar amounts of distearoyl phosphatidylcholine (DSPC) and cholesterol, stable liposomes are obtained. The zeta-potential measurements indicate that both B-6-14- and B-6-16-containing vesicles are negatively charged, with the most negative potential described of any liposome so far. The liposomes are of high potential value as transporters of boron to tumor cells in treatments based on boron neutron capture therapy (BNCT). Liposomes prepared from B-6-14 were slightly less toxic in V79 Chinese hamster cells (IC50 5.6 mM) than unformulated Na2B12H11SH (IC50 3.9 mM), while liposomes prepared from B-6-16 were not toxic even at 30 mM.     

IL-4 increases type 2, but not type 1, cytokine production in CD8+ T cells from mild atopic asthmatics

Background

Virus infections are the major cause of asthma exacerbations. CD8⁺ T cells have an important role in antiviral immune responses and animal studies suggest a role for CD8⁺ T cells in the pathogenesis of virus-induced asthma exacerbations. We have previously shown that the presence of IL-4 during stimulation increases the frequency of IL-5-positive cells and CD30 surface staining in CD8⁺ T cells from healthy, normal subjects. In this study, we investigated whether excess IL-4 during repeated TCR/CD3 stimulation of CD8⁺ T cells from atopic asthmatic subjects alters the balance of type 1/type 2 cytokine production in favour of the latter.

Methods

Peripheral blood CD8⁺ T cells from mild atopic asthmatic subjects were stimulated in vitro with anti-CD3 and IL-2 ± excess IL-4 and the expression of activation and adhesion molecules and type 1 and type 2 cytokine production were assessed.

Results

Surface expression of very late antigen-4 [VLA-4] and LFA-1 was decreased and the production of the type 2 cytokines IL-5 and IL-13 was augmented by the presence of IL-4 during stimulation of CD8⁺ T cells from mild atopic asthmatics.

Conclusion

These data suggest that during a respiratory virus infection activated CD8⁺ T cells from asthmatic subjects may produce excess type 2 cytokines and may contribute to asthma exacerbation by augmenting allergic inflammation.     

Characterization of the interaction of P1,P4-diadenosine 5'-tetraphosphate with luciferase

Adenylated dinucleotides (Ap(n)A) are regulatory molecules that control various cellular processes. A very likely intracellular target for Ap(4)A are enzymes that require ATP as either substrate or modulator. We report the results of new biochemical studies aimed at characterizing the Ap(4)A interaction with firefly luciferase, by using the luminometric and thin layer chromatography techniques. The data presented herein demonstrate that Ap(4)A is a noncompetitive inhibitor for the ATP-induced luminescence. These results together with our previous findings that Ap(4)A is a luciferase substrate [Nucleosides Nucleotides Nucleic Acids 23 (2004) in press.] support the notion that, similar to its interaction with P(2) receptors, Ap(4)A also has a dual interaction with luciferase. Other Ap(n)As (n = 2, 5, and 6) also inhibited the ATP-luciferase interaction. Since Ap(n)As may have similar interactions with other intracellular ATP-requiring enzymes, the study presented herein validates ulterior investigations of the Ap(n)A interaction with such enzymes, and opens the way to a better understanding of their intracellular roles.     

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.     

Isolation and phenotypic characterisation of stem cells from late stage osteoarthritic mesenchymal tissues

Introduction: Osteoarthritis (OA) represents an increasing health issue worldwide. Regenerative medicine (RM) has raised the hope for introducing revolutionary therapies in clinical practice. Detection of autologus cell sources can improve accessibility to RM strategies. Objectives: To assess the presence and biological potential of mesehchymal stem cells in three tissues (subchondral bone, synovial layer, periarticular adipose tissue) in late stages osteoarthritic patients. Material and Methods: Samples were collected from subjects undergoing total knee replacement (TKR). MSCs were isolated and cultured in complete αMEM with β FGF. Cell morphology and growth potential was assessed. Flow cytometry was used for detection of several relevant cell surface markers. Quantitative and qualitative assessment of differentiation potential towards three mesenchymal lineages (osteogenesis adipogenesis chondrogenesis) was performed. Time lapse life cell imaging of nondiferentiated cells over 24 hours period was used to determine cell kinetics. Results: Mesenchymal cells derived from all donors and tissue types showed morphology, growth and surface cell markers associated with stemness. All cell types underwent differentiation toward three mesenchymal lineages with significant differences between tissues of origin, not between donors. Cell kinetics, as derived from life imaging records, was variable with tissue of origin, significant higher for adipose derived MSCS. Conclusion: Human late stage OA mesenchymal tissues, contain progenitors with proliferative and differentiation potential of MSCs. These populations can be used for research and autologus regenerative therapies. Further comparative studies with age matched non OA samples has the potential of contributing to deepening knowledge about disease occurrence and progression.     

Chromatographic retention time prediction for posttranslationally modified peptides

Retention time prediction of peptides in liquid chromatography has proven to be a valuable tool for mass spectrometry-based proteomics, especially in designing more efficient procedures for state-of-the-art targeted workflows. Additionally, accurate retention time predictions can also be used to increase confidence in identifications in shotgun experiments. Despite these obvious benefits, the use of such methods has so far not been extended to (posttranslationally) modified peptides due to the absence of efficient predictors for such peptides. We here therefore describe a new retention time predictor for modified peptides, built on the foundations of our existing Elude algorithm. We evaluated our software by applying it on five types of commonly encountered modifications. Our results show that Elude now yields equally good prediction performances for modified and unmodified peptides, with correlation coefficients between predicted and observed retention times ranging from 0.93 to 0.98 for all the investigated datasets. Furthermore, we show that our predictor handles peptides carrying multiple modifications as well. This latest version of Elude is fully portable to new chromatographic conditions and can readily be applied to other types of posttranslational modifications. Elude is available under the permissive Apache2 open source License at http://per-colator.com or can be run via a web-interface at http://elude.sbc.su.se.