Involvement of caveolin-2 in caveolar biogenesis in MDCK cells

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

A major quantitative trait locus for CD4-CD8 ratio is located on chromosome 11

CD4-CD8 ratio is an important diagnostic measure of immune system functioning. In particular, CD4-CD8 ratio predicts the time taken for progression of HIV infection to acquired immune deficiency syndrome (AIDS) and the long-term survival of AIDS patients. To map genes that regulate differences between healthy individuals in CD4-CD8 ratio, we typed 757 highly polymorphic microsatellite markers at an average spacing of similar to5 cM across the genome in 405 pairs of dizygotic twins at ages 12, 14 and 16. We used multipoint variance components linkage analysis to test for linkage between marker loci and CD4-CD8 ratio at each age. We found suggestive evidence of linkage on chromosome 11p in 12-year-old twins (LOD=2.55, P=0.00031) and even stronger evidence of linkage in the same region at age 14 (LOD 3.51, P=0.00003). Possible candidate genes include CD5 and CD6, which encode cell membrane proteins involved in the positive selection of thymocytes. We also found suggestive evidence of linkage at other areas of the genome including regions on chromosomes 1, 3, 4, 5, 6, 12, 13, 15, 17 and 22.

Epstein-Barr virus-associated Hodgkin's lymphoma

Survivors of Hodgkin's lymphoma (HL) frequently have many years to experience the long-term toxicities of combined modality therapies. Also, a significant proportion of HL patients will relapse or have refractory disease, and less than half of these patients will respond to current salvage strategies. 30–50% of HL cases are Epstein–Barr virus associated (EBV-positive HL). The virus is localized to the malignant cells and is clonal. EBV-positive HL is more frequent in childhood, in older adults (>45 years) and in mixed cellularity cases. The survival of EBV-positive HL in the elderly and the immunosuppressed is particularly poor. Despite improvements in our understanding of EBV-positive HL, the true contribution of EBV to the pathogenesis of HL remains unknown. Increased knowledge of the virus’ role in the basic biology of HL may generate novel therapeutic strategies for EBV-positive HL and the presence of EBV-latent antigens in the malignant HL cells may represent a target for cellular immunotherapy.

IGF-1 phosphorylates AMPK-alpha subunit in ATM-dependent and LKB1-independent manner

Serine/threonine protein kinase AMP-activated protein kinase (AMPK) is a key metabolic stress-responsive factor that promotes the adaptation of cells to their microenvironment. Elevated concentrations of intracellular AMP, caused by metabolic stress, are known to activate AMPK by phosphorylation of the catalytic subunit. Recently, the tumor suppressor serine/threonine protein kinase LKB1 was identified as an upstream kinases, AMPKKs. In the current study, we found that stimulation with growth factors also caused AMPK-alpha subunit phosphorylation. Interestingly, even an LKB1-nonexpressing cancer cell line, HeLa, exhibited growth factor-stimulated AMPK-alpha subunit phosphorylation, suggesting the presence of an LKB1-independent pathway for AMPK-alpha subunit phosphorylation. In the human pancreatic cancer cell line PANC-1, AMPK-alpha subunit phosphorylation promoted by IGF-I was suppressed by antisense ataxia telangiectasia mutated (ATM) expression. We found that IGF-1 also induced AMPK-alpha subunit phosphorylation in the human normal fibroblast TIG103 cell line, but failed to do so in a human fibroblast AT2-KY cell line lacking ATM. Immunoprecipitates of ATM collected from IGF-1-stimulated cells also caused the phosphorylation of the AMPK-alpha subunit in vitro. IGF-1-stimulated ATM phosphorylation at both threonine and tyrosine residues, and our results demonstrated that the phosphorylation of tyrosine in the ATM molecule is important for AMPK-alpha subunit phosphorylation during IGF-1 signaling. These results suggest that IGF-1 induces AMPK-alpha subunit phosphorylation via an ATM-dependent and LKB1-independent pathway. (C) 2004 Elsevier Inc. All rights reserved.

Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides; indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Type 1 nitrergic (ND1) cells of the rabbit retina: Comparison with other axon-bearing amacrine cells

NADPH diaphorase (NADPHd) histochemistry labels two types of nitrergic amacrine cells in the rabbit retina. Both the large ND1 cells and the small ND2 cells stratify in the middle of the inner plexiform layer, and their overlapping processes produce a dense plexus, which makes it difficult to trace the morphology of single cells. The complete morphology of the ND1 amacrine cells has been revealed by injecting Neurobiotin into large round somata in the inner nuclear layer, which resulted in the labelling of amacrine cells whose proximal morphology and stratification matched those of the ND1 cells stained by NADPHd histochemistry. The Neurobiotin-injected ND1 cells showed strong homologous tracer coupling to surrounding ND1 cells, and double-labelling experiments confirmed that these coupled cells showed NADPHd reactivity. The ND1 amacrine cells branch in stratum 3 of the inner plexiform layer, where they produce a sparsely branched dendritic tree of 400-600 mum diameter in ventral peripheral retina. In addition, each cell gives rise to several fine beaded processes, which arise either from a side branch of the dendritic tree or from the tapering of a distal dendrite. These axon-like processes branch successively within the vicinity of the dendritic field before extending, with little or no further branching, for 3-5 mm from the soma in ventral peripheral retina. Consequently, these cells may span one-third of the visual field of each eye, and their spatial extent appears to be greater than that of most other types of axon-bearing amacrine cells injected with Neurobiotin in this study. The morphology and tracer-coupling pattern of the ND1 cells are compared with those of confirmed type 1 catecholaminergic cells, a presumptive type 2 catecholaminergic cell, the type 1 polyaxonal. cells, the long-range amacrine cells, a novel type of axon-bearing cell that also branches in stratum 3, and a type of displaced amacrine cell that may correspond to the type 2 polyaxonal cell. (C) 2004 Wiley-Liss, Inc.

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis. (c) 2005 Wiley Periodicals, Inc.

NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes

Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a C-13-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-C-13] glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using H-1 spin-echo NMR, while C-13[H-1-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.

Discovery of genes associated with fruit ripening in Carica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded: chitinase, 1-aminocyclopropane- 1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species. © 2005 Elsevier Ireland Ltd. All rights reserved.

Homocysteine-lowering treatment with folic acid, cobalamin, and pyridoxine does not reduce blood markers of inflammation, endothelial dysfunction, or hypercoagulability in patients with previous transient ischemic attack or stroke - a randomized substudy

Background and Purpose - Epidemiological and laboratory studies suggest that increasing concentrations of plasma homocysteine ( total homocysteine [tHcy]) accelerate cardiovascular disease by promoting vascular inflammation, endothelial dysfunction, and hypercoagulability. Methods - We conducted a randomized controlled trial in 285 patients with recent transient ischemic attack or stroke to examine the effect of lowering tHcy with folic acid 2 mg, vitamin B-12 0.5 mg, and vitamin B-6 25 mg compared with placebo on laboratory markers of vascular inflammation, endothelial dysfunction, and hypercoagulability. Results - At 6 months after randomization, there was no significant difference in blood concentrations of markers of vascular inflammation (high-sensitivity C-reactive protein [P = 0.32]; soluble CD40L [ P = 0.33]; IL-6 [P = 0.77]), endothelial dysfunction ( vascular cell adhesion molecule-1 [P = 0.27]; intercellular adhesion molecule-1 [P = 0.08]; von Willebrand factor [P = 0.92]), and hypercoagulability (P-selectin [P = 0.33]; prothrombin fragment 1 and 2 [P = 0.81]; D-dimer [P = 0.88]) among patients assigned vitamin therapy compared with placebo despite a 3.7-mumol/L (95% CI, 2.7 to 4.7) reduction in total homocysteine (tHcy). Conclusions - Lowering tHcy by 3.7 mumol/L with folic acid-based multivitamin therapy does not significantly reduce blood concentrations of the biomarkers of inflammation, endothelial dysfunction, or hypercoagulability measured in our study. The possible explanations for our findings are: ( 1) these biomarkers are not sensitive to the effects of lowering tHcy (eg, multiple risk factor interventions may be required); ( 2) elevated tHcy causes cardiovascular disease by mechanisms other than the biomarkers measured; or ( 3) elevated tHcy is a noncausal marker of increased vascular risk.

Degradation of phenanthrene and pyrene in soil slurry reactors with immobilized bacteria Zoogloea sp.

The environmental fate of polycyclic aromatic hydrocarbons (PAHs) in soils is motivated by their wide distribution, high persistence, and potentially deleterious effect on human health. Polycyclic aromatic hydrocarbons constitute the largest group of environmental contaminants released in the environment. Therefore, the potential biodegradation of these compounds is of vital importance. A biocarrier suitable for the colonization by micro-organisms for the purpose of purifying soil contaminated by polycyclic aromatic hydrocarbons was developed. The optimized composition of the biocarrier was polyvinyl alcohol (PVA) 10%, sodium alginate (SA) 0.5%, and powdered activated carbon (PAC) 5%. There was no observable cytotoxicity of biocarriers on immobilized cells and a viable cell population of 1.86 x 10(10) g(-1) was maintained for immobilized bacterium. Biocarriers made from chemical methods had a higher biodegradation but lower mechanical strengths. Immobilized bacterium Zoogloea sp. had an ideal capability of biodegradation for phenanthrene and pyrene over a relative wide concentration range. The study results showed that the biodegradation of phenanthrene and pyrene reached 87.0 and 75.4%, respectively, by using the optimal immobilized method of Zoogloea sp. cultivated in a sterilized soil. Immobilized Zoogloea sp. was found to be effective for biodegrading the soil contaminated with phenanthrene and pyrene. Even in natural (unsterilized) soil, the biodegradation of phenanthrene and pyrene using immobilized Zoogloea sp. reached 85.0 and 67.1%, respectively, after 168 h of cultivation, more than twice that achieved if the cells were not immobilized on the biocarrier. Therefore, the immobilization technology enhanced the competitive ability of introduced micro-organisms and represents an effective method for the biotreatment of soil contaminated with phenanthrene and pyrene.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Plasminogen activator system in osteoclasts

To determine which genes of the plasminogen activator (PA) system were expressed in osteoclasts, RNA extracted from microisolated mouse osteoclasts was used as template for reverse transcribed polymerase chain reaction (RT-PCR) with gene-specific primer pairs, Using this approach, the expression of RNAs for tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, protease nexin, and urokinase receptor isoform 1 (uPAR1) were detected in mouse osteoclasts. The expression of uPAR RNA in osteoclasts was confirmed by in situ hybridization with a uPAR1 probe, RNA encoding the uPAR isoform 2 was not detected in mouse osteoclasts, but a novel unspliced uPAR RNA variant was detected in these cells, The novel uPAR variant and uPAR1 RNA were also detected in mouse calvarial osteoblasts, kidney, muscle, and the mouse macrophage cell line J774A.1 by RT-PCR The presence of RNAs for most of the components of the PA system in osteoclasts suggests that it may have a functional role in this cell type.

Tubular eyes of deep-sea fishes: A comparative study of retinal topography

The world's deep oceans are home to a number of teleosts with asymmetrical or tubular eyes. These immobile eyes possess large spherical lenses and subtend a large binocular visual field directed either dorsally or rostrally. Derived from a lateral non-tubular eye, the tubular eye is comprised of a thick main retina, subserving the rostrally or dorsally directed binocular visual field, and a thin accessory retina subserving, the lateral, monocular visual field. The main retina is thought to receive a focussed image, while the accessory retina is too close to the lens for a focussed image to be received. Several species also possess retinal diverticula, which are small evaginations of differentiated retina located in the rostrolateral wall of the eye and thought to increase the visual field. In order to investigate the spatial resolving power of these retinae (main, accessory and diverticulum), the distribution of cells within the ganglion cell layer was analysed from retinal wholemounts and sectioned material in ten species representing four genera. In all species, the main retina possesses a marked increase in cell density towards a specialised retinal region (area centralis), with a centro-peripheral gradient range between 7.1 and 60:1 and a peak density range of between 30 and 55 x 10(3) cells per mm(2). The accessory retinae and the transitional zone between the main and accessory retinae possess relatively low cell densities (between 1 and 10 x 10(3) cells per mm(2)) and lack an area centralis. Retinal diverticula examined in four species possess mean ganglion cell densities of between 7.2 and 109.4 x 10(3) cells per mm(2). Analyses of soma areas show that the ganglion cell layer of most species possesses cells with areas in a range of 8.0 to 15.4 mu m(2) in the main retina and between 15.1 and 17.4 mu m(2) in the accessory retina. The peak spatial resolving power of the main retina of the ten species varies from 4.1 to 9.1 cycles per degree. The positions of the retinal areae centrales relative to each species' binocular visual field are discussed in relation to what is known of feeding behaviour of these fishes in the deep-sea.