Identification of 8-iso-prostaglandin F(2alpha) in rat brain neuronal endings: a possible marker of membrane phospholipid peroxidation

Isoprostanes are a family of prostaglandin (PG) F and E isomers generated by free-radical attack from membrane bound arachidonic acid. We measured detectable levels of 8-iso-PGF(2alpha) in the perfusates of synaptosomes obtained from different areas of the rat brain cortex. A small but significant release of this isoprostane was found under basal conditions from all the areas explored; being lower in the dorsal cortex in respect to the frontal, parietal and occipital areas. Exposure of synaptosomes to a phospholipase A(2) activator, i.e. calcium-ionophore A23187, an oxidant agent, such as hydrogen peroxide or amyloid beta-peptide did not modify 8-iso-PGF(2alpha) release when these stimuli were applied separately. However, either hydrogen peroxide or amyloid beta-peptide increased 8-iso-PGF(2alpha) release in a dose-dependent manner, when given in the presence of the calcium-ionophore A23187. Synaptosome treatment with a non-selective cyclooxygenase inhibitor (fenoprofen) did not modify 8-iso-PGF(2alpha) release in any way, but treatment with a water soluble antioxidant (Trolox C) completely suppressed isoprostane release under basal conditions, as well as after the oxidant injury induced either by hydrogen peroxide or amyloid beta-peptide. We conclude that, in neuronal endings, 8-iso-PGF(2alpha) is generated under basal conditions and its formation may be increased in a dose-dependent fashion by oxidant stimuli through a cyclooxygenase-independent mechanism involving free radical-catalyzed oxidation of arachidonic acid on membrane phospholipids.     

Cytogenetic mapping of five YAC clones to human chromosome region 2q31 --> q32.1 in relation to the FRA2G common fragile site

In this work, five YAC clones have been mapped by fluorescent in situ hybridization (FISH) to human chromosome region 2q31 q32.1 and ordered in relation to each other and to the FRA2G common fragile site. YAC clones that span the fragile site have been identified. Moreover a deleted HOXD 13 gene has been identified on the 942D2 YAC.     

Absence of the CD1 molecule up-regulates antitumor activity induced by CpG oligodeoxynucleotides in mice

The role of NKT cells on antitumor activity of CpG oligodeoxynucleotides (ODNs) was evaluated by peritumoral injections of CpG-ODNs in s.c. melanoma-bearing mice of strains differing in the number of NKT cells (athymic nude mice, recombination-activating gene(-/-)/transgenic V(alpha)14/Vbeta8.2 mice that generate NKT cells; J(alpha)281(-/-) mice and CD1(-/-) mice, which both have a strongly reduced number of NKT cells; and C57BL/6 wild-type mice). Tumor growth was significantly inhibited in strains enriched or depleted of NKT cells. The two murine strains having a reduced number of NKT cells differed significantly in the CpG-dependent tumor growth inhibition: in J(alpha)281(-/-) mice this inhibition was superimposable to that observed in C57BL/6 mice, while in CD1(-/-) mice the inhibition was dramatic. The increased tumor inhibition in CD1(-/-) correlated with a significantly higher ratio of IFN-gamma-IL-4 production in response to CpG as compared with C57BL/6 and J(alpha)281(-/-) mice. Experiments in which preparations of APCs and lymphocytes of the three strains were mixed showed that in the presence of APCs not expressing CD1, the production of CpG-ODN-induced type 1 cytokines was higher. Phenotype analysis of IFN-gamma- and IL-4-producing cells revealed that the differences between CD1(-/-) and C57BL/6 in the production of these two cytokines were mainly due to CD3(+) T lymphocytes. These data point to a regulatory role for the CD1 molecule in antitumor activity induced by danger signals, independently of V(alpha)14 NKT cells. The identification of a CD1-dependent suppressive subpopulation(s) might have important implications for the study of tolerance in the context of cancer, autoimmunity, and transplantation.     

Both vegetative and reproductive actin isovariants complement the stunted root hair phenotype of the Arabidopsis act2-1 mutation

The ACT2 gene, encoding one of eight actin isovariants in Arabidopsis, is the most strongly expressed actin gene in vegetative tissues. A search was conducted for physical defects in act2-1 mutant plants to account for their reduced fitness compared with wild type in population studies. The act2-1 insertion fully disrupted expression of ACT2 RNA and significantly lowered the level of total actin protein in vegetative organs. The root hairs of the act2-1 mutants were 10% to 70% the length of wild-type root hairs, and they bulged severely at the base. The length of the mutant root hairs and degree of bulging at the base were affected by adjusting the osmolarity and gelling agent of the growth medium. The act2-1 mutant phenotypes were fully rescued by an ACT2 genomic transgene. When the act2-1 mutation was combined with another vegetative actin mutation, act7-1, the resulting double mutant exhibited extensive synergistic phenotypes ranging from developmental lethality to severe dwarfism. Transgenic overexpression of the ACT7 vegetative isovariant and ectopic expression of the ACT1 reproductive actin isovariant also rescued the root hair elongation defects of the act2-1 mutant. These results suggest normal ACT2 gene regulation is essential to proper root hair elongation and that even minor differences may cause root defects. However, differences in the actin protein isovariant are not significant to root hair elongation, in sharp contrast to recent reports on the functional nonequivalency of plant actin isovariants. Impairment of root hair functions such as nutrient mining, water uptake, and physical anchoring are the likely cause of the reduced fitness seen for act2-1 mutants in multigenerational studies.     

Chemiosmotic ATP synthesis in photosynthetically inactive chromoplasts from Narcissus pseudonarcissus L. linked to a redox pathway potentially also involved in carotene desaturation

Mature chromoplasts from daffodil (Narcissus pseudonarcissus) flowers, although devoid of thylakoid structures, contain immunologically detectable alpha-subunits of ATP-synthase (H(+)-transporting ATP phosphohydrolase; EC 3.6.3.14). To show the presence of the entire functional protein complex, chromoplast membrane proteins were solubilized and reconstituted in phosphatidylcholine liposomes. The membranes were energized by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential, and the initial rate of ATP synthesis was measured with a luciferin/luciferase assay. In addition, by demonstrating NADPH-dependent ATP synthesis, we show that an NAD(P)H-dependent respiratory redox pathway in chromoplasts, previously identified as an important constituent of the carotene desaturation system, proceeds concomitant with membrane energization.     

Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms

Complex glycoinositolphosphoryl ceramides (GIPCs) have been purified from a pathogenic encapsulated wild-type (WT) strain of Cryptococcus neoformans var. neoformans and from an acapsular mutant (Cap67). The structures of the GIPCs were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance spectroscopy, methylation analysis, gas chromatography-mass spectrometry, and chemical degradation. The main GIPC from the WT strain had the structure Manp(alpha1-3)[Xylp(beta1-2)] Manp(alpha1-4)Galp(beta1-6)Manp(alpha1-2)Ins-1-phosphoryl ceramide (GIPC A), whereas the compounds from the acapsular mutant were more heterogeneous in their glycan chains, and variants with Manp(alpha1-6) (GIPC B), Manp(alpha1-6) Manp(alpha1-6) (GIPC C), and Manp(alpha1-2)Manp(alpha1-6)Manp(alpha1-6) (GIPC D) substituents linked to the nonreducing terminal mannose residue found in the WT GIPC A were abundant. The ceramide moieties of C. neoformans GIPCs were composed of a C(18) phytosphingosine long-chain base mainly N-acylated with 2-hydroxy-tetracosanoic acid in the WT GIPC while in the acapsular Cap67 mutant GIPCs, as well as 2-hydroxy-tetracosanoic acid, the unusual 2,3-dihydroxy-tetracosanoic acid was characterized. In addition, structural analysis revealed that the amount of GIPC in the WT cells was fourfold less of that in the acapsular mutant.     

Chemoprevention strategies in the prostate: an overview

Chemoprevention is the administration of agents (drugs, biologics, and nutrients) to prevent induction, inhibit, or delay the progression of cancers. Prostate cancer is an important target for chemoprevention because of its long latency and high prevalence. The development of rational chemopreventive strategies requires knowledge of the mechanisms of prostate carcinogenesis and identification of agents that interfere with these mechanisms. Because of the long time period for prostate carcinogenesis and the large size of the cohort required for an evaluable study, identification and characterization of early intermediate biomarkers and their validation as surrogate endpoints for cancer incidence are essential for chemopreventive agent development. Finally, suitable populations with appropriate risk factors, including the presence of premalignant lesions and genetic predispositions, need to be well characterized for future chemopreventive interventions.     

UVA light stimulates the production of cathepsin G and elastase-like enzymes by dermal fibroblasts: a possible contribution to the remodeling of elastotic areas in sun-damaged skin

Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.