Increased oxidative stress in the RAW 264.7 macrophage cell line is partially mediated via the S-nitrosothiol-induced inhibition of glutathione reductase

We investigated whether endogenously or exogenously produced nitric oxide (NO) can inhibit cellular glutathione reductase (GR) via the formation of S-nitrosothiols to decrease cellular glutathione (GSH) and increase oxidative stress in RAW 264.7 cells. The specificity of this inhibition was demonstrated by addition of a NO-synthase inhibitor, and met- or oxyhemoglobin. Using isolated GR we found that only certain NO donors inhibit this enzyme via S-nitrosothiol. Furthermore, we found that cellular GSH decrease is paralleled by an increase of superoxide anion production. Our results show that the GR enzyme is a potential target of S-nitrosothiols to decrease cellular GSH levels and to induce oxidative stress in macrophages.     

Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var. capitata)

We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage. Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa. The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively. Expression of this cDNA clone in E. coli shows a functional PLD activity similar to that of the natural PLD.     

A novel reactive perstraction system based on liquid-core microcapsules applied to lipase-catalyzed biotransformations

A novel reactive perstraction system has been developed based on liquid-core capsules, involving an enzyme-catalyzed reaction coupled with simultaneous in situ product recovery. Lipase-catalyzed reactions, hydrolysis of triprionin and nitrophenyl laurate, were selected to test the system and demonstrate the feasibility of immobilization of enzymes to the membranes of liquid-core capsules and the ability to extract hydrophobic products of the reaction within the capsule core. The lipase from Candida rugosa was immobilized to the microcapsules by adsorption and by covalent binding through activation with glutaraldehyde. In both cases improved temperature and operational stability were achieved. Both types of immobilization resulted in a basic shift of the pH optimum for activity, from 7.5 to 9.0. The presence of an organic phase within the capsule core allowed direct product separation and lead to a decrease in product inhibition of the lipase-catalyzed reaction.     

Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA

We have used oligonucleotides made of 2'-OMe RNA to analyze the role of separate domains of U2 snRNA in the splicing process. We show that antisense 2'-OMe RNA oligonucleotides bind efficiently and specifically to U2 snRNP and demonstrate that masking of two separate regions of U2 snRNA can inhibit splicing by affecting different steps in the spliceosome assembly pathway. Masking the 5' terminus of U2 snRNA does not prevent U2 snRNP binding to pre-mRNA but blocks subsequent assembly of a functional spliceosome. By contrast, masking of U2 sequences complementary to the pre-mRNA branch site completely inhibits binding of pre-mRNA. Hybrid formation at the branch site complementary region also triggers a specific change which affects the 5' terminus of U2 snRNA.     

Protective effect of CardiPro against doxorubicin-induced cardiotoxicity in mice

The effect of CardiPro, a polyherbal formulation, with an antioxidant property, has been studied on doxorubicin (DXR)-induced cardiotoxicity in mice. CardiPro (150 mg/kg b.w., twice daily was administered orally for 7 weeks along with four equal injections (each containing 4.0 mg/kg b.w., DXR) intraperitoneally, once weekly (cumulative dose 16 mg/kg). After a 3-week post DXR treatment period, cardiotoxicity was assessed by noting mortality, volume of ascites, liver congestion, changes in heart weight, myocardial lipid peroxidation, antioxidant enzymes and histology of heart. DXR-treated animals showed higher mortality (50%) and more ascites. Myocardial SOD and glutathione peroxidase activity were decreased and lipid peroxidation was increased. Histology of heart of DXR-treated animals showed loss of myofibrils and focal cytoplasmic vacuolization. CardiPro significantly protected the mice from DXR-induced cardiotoxic effects as evidenced by lower mortality (25%), less ascites, myocardial lipid peroxidation, normalization of antioxidant enzymes and minimal damage to the heart histologically. Our data confirm the earlier reports that DXR cardiotoxicity is associated with the free radical-induced tissue damage. Administration of CardiPro, with an antioxidant property, protected the DXR-induced cardiotoxicity in mice.     

Genetic differentiation in relation to marine landscape in a broadcast-spawning bivalve mollusc (Placopecten magellanicus)

Marine bivalves are sessile or sedentary as adults but have planktonic larvae which can potentially disperse over large distances. Consequently larval transport is expected to play a prominent role in facilitating gene flow and determining population structure. The sea scallop (Placopecten magellanicus) is a dioecious species with high fecundity, broadcast spawning and a c. 30-day planktonic larval stage, yet it forms discrete populations or 'beds' which have significantly different dynamics and characteristics. We analysed variation at six microsatellite loci in 12 locations throughout the geographic range of the species from Newfoundland, Canada, to New Jersey, USA. Significant differentiation was present and the maximum pairwise theta value, between one of the Newfoundland samples in the north and a sample from the southern portion of the range, was high at 0.061. Other proximate pairs of samples had no detectable genetic differentiation. Mantel tests indicated a significant isolation by distance, but only when one of the populations was excluded. A landscape genetic approach was used to detect areas of low gene flow using a joint analysis of spatial and genetic information. The two major putative barriers inferred by Monmonier's algorithm were then used to define regions for an analysis of molecular variance (amova). That analysis showed a significant but low percentage (1.2%) of the variation to be partitioned among regions, negligible variation among populations within regions, and the majority of the variance distributed between individuals within populations. Prominent currents were concordant with the demarcation of the regions, while a novel approach of using particle tracking software to mimic scallop larval dispersal was employed to interpret within-region genetic patterns.     

Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-sepharose chromatography

Two forms of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from both bovine fetal skin and calf articular cartilage and characterized. The proteoglycans were isolated using either (a) molecular sieve chromatography under conditions where DS-PGI selectively self-associates or (b) chromatography on octyl-Sepharose, which separates DS-PGI from DS-PGII based on differences in the hydrophobic properties of their core proteins. The NH2-terminal amino acid sequence of DS-PGI from skin and cartilage is identical. The NH2-terminal amino acid sequence of DS-PGII from skin and cartilage is identical. However, the amino acid sequence data and tryptic peptide maps demonstrate that the core proteins of DS-PGI and DS-PGII differ in primary structure. In DS-PGI from bovine fetal skin, 81-84% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4) disaccharide repeating units. In DS-PGI from calf articular cartilage, only 25-29% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4). In DS-PGII from bovine fetal skin, 85-93% of the glycosaminoglycan was IdoA-GalNAc(SO4), whereas in DS-PGII from calf articular cartilage, only 40-44% of the glycosaminoglycan was IdoA-GalNAc(SO4). Thus, analogous proteoglycans from two different tissues, such as DS-PGI from skin and cartilage, possess a core protein with the same primary structure, yet contain glycosaminoglycan chains which differ greatly in iduronic acid content. These differences in the composition of the glycosaminoglycan chains must be determined by tissue-specific mechanisms which regulate the degree of epimerization of GlcA-GalNAc(SO4) into IdoA-GalNAc(SO4) and not by the primary structure of the core protein.     

Adrenergic origin of very low-frequency blood pressure oscillations in the unanesthetized rat

Spectral analysis of cardiovascular signals has been extensively used to investigate circulatory homeostatic mechanisms. However, the nature of very low-frequency (VLF) fluctuations remains unclear. Because we previously observed enhanced VLF fluctuations in blood pressure (BP) in the sympathectomized rat (a model characterized by markedly increased plasma epinephrine levels), the aims of our study were to assess whether the genesis of VLF fluctuations in BP depends on circulating catecholamines and to determine which adrenergic receptor(s) and which membrane ion channel(s) are involved. We used continuous intra-arterial BP recordings from unanesthetized unrestrained rats to compute the power of VLF fluctuations in BP in the intact condition, during acute ganglionic blockade with hexamethonium, and after restoration of BP levels by infusion (in addition to hexamethonium) of adrenergic agonists (epinephrine, norepinephrine, and clonidine) or nonadrenergic vasoconstrictors (vasopressin). Effects of infusion of specific adrenergic receptor blockers (propranolol, prazosin, and yohimbine) with hexamethonium and catecholamines and infusion of various membrane ion channel blockers on VLF fluctuations in BP were also evaluated. Our results are as follows. 1) Ganglionic blockade drastically reduced BP levels and VLF fluctuations. 2) All vasoconstrictors restored BP levels, but only adrenergic vasoconstrictors generated striking VLF fluctuations in BP. 3) Catecholamine-induced fluctuations were abolished by alpha2-, but not alpha1- or beta-, adrenergic receptor blockade and by Ba2+-sensitive K+ channel or L-type Ca2+ channel, but not by other ion channel, blockers. We conclude that, in the conscious, unrestrained ganglion-blocked rat, catecholamine infusion generates VLF fluctuations in BP through stimulation of alpha2-receptors and activation of Ba2+-sensitive K+ channels. These fluctuations may have (patho)physiological relevance under conditions of disrupted circulatory homeostasis.     

The role of macrophages in B cell tolerance. I. Antigen-specific failure of Thy-1, Ly-2 negative adherent cells from tolerant mice to reconstitute immunocompetence in adherent cell deficient spleen cells

T-Cell-independent B-cell tolerance to the hapten derivatives of carboxymethyl cellulose (CMC) or methyl cellulose (MC) appears to be controlled by Thy-1-, Ly-2- adherent (A) cells contained in the spleen or peritoneal fluid. Immunocompetence in nonadherent (NA) normal spleen cells could be restored in vitro by irradiated A cells from normal mice. However, NA cells reconstituted with irradiated A cells derived from hapten specifically tolerant mice failed to respond to the same hapten, but responded normally to an immunogenic challenge with another unrelated antigen. A cells that had been preincubated at 4 degrees C with hapten derivatized MC also failed to restore immunocompetence. While preincubation of unfractionated spleen cells with the tolerogen under the same conditions resulted in B-cell unresponsiveness, such treatment of NA cells failed to render B cells tolerant. Treatment of A cells from tolerant mice with the reducing agent potassium iodide (KI) in vitro restored their capacity to render cultures of NA cells immunocompetent to the relevant hapten. Moreover, treatment with KI of spleen cells from mice injected with the tolerogen was shown to render them responsive. We suggest that B-cell tolerance induced by hapten derivatives of CMC and MC is mediated by suppressive macrophages contained among A cells. Certain subpopulations of macrophages are known to exert cytotoxic effects upon target cells by the release at close range of oxidating agents. We postulate that hapten derivatized CMC and MC, through unique properties of the carrier, bind to and possibly activate macrophages rendering them specifically suppressive for hapten binding B cells.