Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycaemic conditions.

The neuronal effects of glucose deficiency on amino acid metabolism was studied on three-dimensional cultures of rat telencephalon neurones. Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone-specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult. Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose-deficiency. Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased. Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage. Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage. Both lactate and glutamine were readily oxidized in these neuronal cultures. The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions. This results in increased ammonia production and neuronal damage.

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) but not PPARalpha serves as a plasma free fatty acid sensor in liver.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARalpha target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARalpha, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARbeta/delta during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARalpha. Binding of PPARbeta/delta to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARbeta/delta by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARbeta/delta. In contrast, the data did not support activation of PPARalpha by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARbeta/delta target genes and show that upregulation of gene expression by PPARbeta/delta is sensitive to plasma FFA levels. In contrast, this is not the case for PPARalpha, revealing a novel mechanism for functional differentiation between PPARs.

Total lipid and fatty acid accumulation during basidiospore formation in the ectomycorrhizal fungus Pisolithus sp.

The basidiospores of Pisolithus sp. contain large amounts of lipids, indicating provision for future germination in the host rhizosphere. However, the accumulation, composition, and mobilization of lipids during formation and germination of these spores are largely unknown. In this study, lipid storage and fatty acid composition during basidiosporogenesis were analyzed in fresh basidiocarps using bright-field microscopy and gas chromatography. Abundant lipid bodies are found in the hyphae, basidia, and basidiospores of fungal basidiocarps. This evidences a considerable C transport in the basidiocarp to meet the C demand during basidiospore formation. Fatty acid composition analysis revealed the presence of 24 compounds with chains of 9 to 18 C atoms, either saturated or insaturated, with one or two insaturations. The fatty acid composition and content varied according to the developmental stage of the peridioles. In free basidiospores, the predominant compounds were 16:0, 16:1w5c, 18:1w9c, and 18:2w6,9c/18:0ante, at concentrations of 76, 46, 192, and 51 µg g-1 dry matter, respectively. Our results indicate that oleic acid is the major constituent of lipid reserves in Pisolithus sp. basidiospores. Further studies are being conducted to determine the factors that induce lipid mobilization during spore germination.

Influence of the timing of low-dose aspirin on tolerability of prolonged-release nicotinic acid in patients at elevated cardiovascular risk

OBJECTIVES: To investigate the effect of low-dose aspirin administered in the morning or evening on the rate of discontinuation of prolonged-release nicotinic acid (Niaspan) due to flushing in patients at elevated cardiovascular risk. RESEARCH DESIGN AND METHODS: This was an observational, non-interventional study in patients at elevated cardiovascular risk due to cardiovascular disease or type 2 diabetes. Patients received prolonged-release nicotinic acid and aspirin under the usual care of their physician for 15 weeks. MAIN OUTCOME MEASURES: The main outcome measure was the rate of treatment discontinuation for flushing. Other adverse drug reactions (ADRs) were also recorded. Lipid parameters were also measured. RESULTS: The patient population included 539 subjects (70% male); 36% had type 2 diabetes, 80% had prior cardiovascular disease, and 37% had a family history of cardiovascular disease. The rate of treatment discontinuation due to flushing did not differ (p = 0.3375) between the morning aspirin group (10.6%) and the evening aspirin group (13.8%). The overall incidence of flushing was 57%. Most flushes were of mild or moderate severity and decreases occurred over time in both frequency and intensity. ADRs unrelated to flushing occurred in 6.6% of the morning aspirin group and 7.4% of the evening aspirin group. HDL-cholesterol increased by +21.3% in the overall population, together with moderate improvements in other lipid parameters. CONCLUSIONS: Flushing was the most common ADR with prolonged-release nicotinic acid treatment, as expected. The timing of aspirin administration did not influence the rate of treatment discontinuations for flushing. Marked increases in HDL-cholesterol were observed.

α-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism.

Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical α-ketoglutarate (αKG) transport in the proximal tubule and Henle's loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of αKG are sensed by the αKG receptor OXGR1 expressed in the type B and non-A-non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM αKG to the tubular lumen strongly stimulated Cl--dependent HCO3- secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1-/- mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1-/- mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO3- secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish αKG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.

Multicopy suppression of a gacA mutation by the infC operon in Pseudomonas fluorescens CHA0: competition with the global translational regulator RsmA.

The gacA gene of the biocontrol strain Pseudomonas fluorescens CHA0 codes for a response regulator which, together with the sensor kinase GacS (=LemA), is required for the production of exoenzymes and secondary metabolites involved in biocontrol, including hydrogen cyanide (HCN). A gacA multicopy suppressor was isolated from a cosmid library of strain CHA0 and identified as the infC-rpmI-rplT operon, which encodes the translation initiation factor IF3 and the ribosomal proteins L35 and L20. The efficiency of suppression was about 30%, as determined by the use of a GacA-controlled reporter construct, i.e. a translational hcnA'-'lacZ fusion. Overexpression of the rsmA gene (coding for a global translational repressor) reversed the suppressive effect of the amplified infC operon. This finding suggests that some product(s) of the infC operon can compete with RsmA at the level of translation in P. fluorescens CHA0 and that important biocontrol traits can be regulated at this level.

Cloning of an interferon-gamma regulated human melanoma-associated antigen: identity to the intercellular adhesion molecule ICAM-1.

The human melanoma-associated antigen identified by the monoclonal antibody (mAb) Me14-D12 is a cell surface protein whose expression is induced by interferon-gamma (IFN-gamma). We have recently reported the molecular cloning of a genomic probe specific for the gene and mRNA of this protein. By screening with the genomic probe, we have now isolated a full length 3.0 kb cDNA from a Raji cell line-derived lambda-gt10 library. Sequence analysis of this cDNA showed a 99.8% homology with the intercellular adhesion molecule-1 (ICAM-1). Mouse Ltk- cells stably transfected with the human cDNA clone were found to express the ICAM-1 antigenic determinants detected by mAb Me14-D12 and a reference anti-ICAM-1 mAb, as judged by surface immunofluorescence. Immunoprecipitation of surface-iodinated proteins with mAb Me14-D12 revealed the presence of a 90 kD molecule with identical mobility to ICAM-1. In addition, mAb Me14-D12 could inhibit the phorbolester-stimulated aggregation of U937 cells. The findings show that the human melanoma-associated Me14-D12 antigen is the adhesion molecule ICAM-1.

An unusual uterine tumour with signet ring cell features

Objective: Lymphomas with signet ring cell features are rare, as is uterine dissemination of lymphomas. We report an exceptional case of a uterine tumor combining these two characteristics. Method: A 61-year-old female was diagnosed in 2004 with localized nodal grade 2 follicular lymphoma (stage IA). She received local radiation therapy, experienced total remission, and did well until 2009 when a systematic CT scan evidenced a pelvic anterior-lateral mass. Total enlarged hysterectomy was performed. Results: The anterior uterine wall contained a 4.8-cm fish flesh well-delineated mass corresponding to a mostly diffuse and focally nodular proliferation of medium to large cells with extensive signet ring cell changes. Tumor cells were CD20-, CD10-, Bcl2-, and Bcl6-positive with a low proliferation rate (<10-15%); CD21 underlined a focal follicular architecture. The vacuoles were PAS-negative and did not stain for immunoglobulin; ultrastructural analysis revealed nonspecific degenerative vacuoles. No lymph nodes were identified isolated from the surgical specimen. The tumor was considered as a secondary localization of the systemic follicular lymphoma, though no signet ring cells were evidenced in the cervical lymph node biopsy (reviewed). Follow-up showed retroperitoneal tissue infiltration (PET-CT) and normal medullar biopsy. She recently started R-CHOP chemotherapy. Conclusion: This case illustrates both an unusual site of dissemination and challenging cytological characteristics in a follicular lymphoma. The signet ring cell changes challenged the adequate classification of this lymphoma as either a large B cell or a follicular B cell lymphoma.

Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.

The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.

Structural and gas-sensing properties of WO3 nanocrystalline powders obtained by a sol-gel method from tungstic acid

WO3 nanocrystalline powders were obtained from tungstic acid following a sol-gel process. Evolution of structural properties with annealing temperature was studied by X-ray diffraction and Raman spectroscopy. These structural properties were compared with those of WO3 nanopowders obtained by the most common process of pyrolysis of ammonium paratungstate, usually used in gas sensors applications. Sol-gel WO3 showed a high sensor response to NO2 and low response to CO and CH4. The response of these sensor devices was compared with that of WO3 obtained from pyrolysis, showing the latter a worse sensor response to NO2. Influence of operating temperature, humidity, and film thickness on NO2 detection was studied in order to improve the sensing conditions to this gas.

The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription.

The transcriptional transactivational activities of the phosphoprotein cAMP-response element-binding protein (CREB) are activated by the cAMP-dependent protein kinase A signaling pathway. Dimers of CREB bind to the palindromic DNA element 5'-TGACGTCA-3' (or similar motifs) called cAMP-responsive enhancers (CREs) found in the control regions of many genes, and activate transcription in response to phosphorylation of CREB by protein kinase A. Earlier we reported on the cyclical expression of the CREB gene in the Sertoli cells of the rat testis that occurred concomitant with the FSH-induced rise in cellular cAMP levels and suggested that transcription of the CREB gene may be autoregulated by cAMP-dependent transcriptional proteins. We now report the structure of the 5'-flanking sequence of the human CREB gene containing promoter activity. The promoter has a high content of guanosines and cytosines and lacks canonical TATA and CCAAT boxes typically found in the promoters of genes in eukaryotes. Notably, the promoter contains three CREs and transcriptional activities of a promoter-luciferase reporter plasmid transfected to placental JEG-3 cells are increased 3- to 5-fold over basal activities in response to either cAMP or 12-O-tetradecanoyl phorbol-14-acetate, and give 6- to 7-fold responses when both agents are added. The CREs bind recombinant CREB and endogenous CREB or CREB-like proteins contained in placental JEG-3 cells and also confer cAMP-inducible transcriptional activation to a heterologous minimal promoter. Our studies suggest that the expression of the CREB gene is positively autoregulated in trans.

Expression analyses of three members of the AtPHO1 family reveal differential interactions between signaling pathways involved in phosphate deficiency and the responses to auxin, cytokinin, and abscisic acid.

The PHO1 protein is involved in loading inorganic phosphate (Pi) to the root xylem. Ten genes homologous to AtPHO1 are present in the Arabidopsis thaliana (L.) Heyn genome. From this gene family, transcript levels of only AtPHO1, AtPHO1;H1 and AtPHO1;H10 were increased by Pi-deficiency. While the up-regulation of AtPHO1;H1 and AtPHO1;H10 by Pi deficiency followed the same rapid kinetics and was dependent on the PHR1 transcription factor, phosphite only strongly suppressed the expression of AtPHO1;H1 and had a minor effect on AtPHO1;H10. Addition of sucrose was found to increase transcript levels of both AtPHO1 and AtPHO1;H1 in Pi-sufficient or Pi-deficient plants, but to suppress AtPHO1:H10 under the same conditions. Treatments of plants with auxin or cytokinin had contrasting effect depending on the gene and on the Pi status of the plants. Thus, while both hormones down-regulated expression of AtPHO1 independently of the plant Pi status, auxin and cytokinin up-regulated AtPHO1;H1 and AtPHO1;H10 expression in Pi-sufficient plants and down-regulated expression in Pi-deficient plants. Treatments with abscisic acid inhibited AtPHO1 and AtPHO1;H1 expression in both Pi-sufficient and Pi-deficient plants, but increased AtPHO1;H10 expression under the same conditions. The inhibition of expression by abscisic acid of AtPHO1 and AtPHO1;H1, and of the Pi-starvation responsive genes AtPHT1;1 and AtIPS1, was dependant on the ABI1 type 2C protein phosphatase. These results reveal that various levels of cross talk between the signal transduction pathways to Pi, sucrose and phytohormones are involved in the regulation of expression of the three AtPHO1 homologues.