Upregulation of Neuronal Nitric Oxide Synthase in in Vitro Stellate Astrocytes and in Vivo Reactive Astrocytes After Electrically Induced Status Epilepticus

Neuronal nitric oxide synthase (nNOS) is a constitutively expressed and calcium-dependent enzyme. Despite predominantly expressed in neurons, nNOS has been also found in astrocytes, although at lower expression levels. We have studied the regulation of nNOS expression in cultured rat astrocytes from cortex and spinal cord by Western blotting and immunocytochemistry. nNOS was not detectable in cultured astrocytes grown in serum-containing medium (SCM), but was highly expressed after serum deprivation. Accordingly, calcium-dependent NOS activity and both intracellular nitrite levels and nitrotyrosine immunoreactivity after glutamate stimulation were higher in serum-deprived astrocytes than in cells grown in SCM. Serum deprivation induced a modification of astrocytes morphology, from flat to stellate. nNOS upregulation was also observed in reactive astrocytes of rat hippocampi after electrically induced status epilepticus, as demonstrated by double-labeling experiments. Thus, nNOS upregulation occurs in both in vitro stellate and in vivo reactive astrocytes, suggesting a possible involvement of glial nNOS in neurological diseases characterized by reactive gliosis.     

The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways

The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [(3)H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but (3)H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1.     

Functional characterization and crystal structure of the C215D mutant of protein-tyrosine phosphatase-1B

We have characterized the C215D active-site mutant of protein-tyrosine phosphatase-1B (PTP-1B) and solved the crystal structure of the catalytic domain of the apoenzyme to a resolution of 1.6 A. The mutant enzyme displayed maximal catalytic activity at pH approximately 4.5, which is significantly lower than the pH optimum of 6 for wild-type PTP-1B. Although both forms of the enzyme exhibited identical Km values for hydrolysis of p-nitrophenyl phosphate at pH 4.5 and 6, the kcat values of C215D were approximately 70- and approximately 7000-fold lower than those of wild-type PTP-1B, respectively. Arrhenius plots revealed that the mutant and wild-type enzymes displayed activation energies of 61 +/- 1 and 18 +/- 2 kJ/mol, respectively, at their pH optima. Unlike wild-type PTP-1B, C215D-mediated p-nitrophenyl phosphate hydrolysis was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane, suggesting a direct involvement of Asp215 in catalysis. Increasing solvent microviscosity with sucrose (up to 40% (w/v)) caused a significant decrease in kcat/Km of the wild-type enzyme, but did not alter the catalytic efficiency of the mutant protein. Structurally, the apoenzyme was identical to wild-type PTP-1B, aside from the flexible WPD loop region, which was in both "open" and "closed" conformations. At physiological pH, the C215D mutant of PTP-1B should be an effective substrate-trapping mutant that can be used to identify cellular substrates of PTP-1B. In addition, because of its insensitivity to oxidation, this mutant may be used for screening fermentation broth and other natural products to identify inhibitors of PTP-1B.     

Single-sample preparation for simultaneous cellular redox and energy state determination

A simple and reliable method for the preparation of biological samples for the evaluation of biochemical parameters representative of the redox and energy states, such as glutathione (GSH), oxidized glutathione (GSSG), oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH), coenzyme A (CoASH), oxidized CoASH, ascorbate, malondialdehyde, oxypurines, nucleosides, and energy metabolites, is presented. Fast deproteinization under nonoxidizing conditions is obtained by tissue homogenization in ice-cold, nitrogen-saturated CH3CN + 10 mM KH2PO4 (3:1; v:v), pH 7.40. After sample centrifugation to pellet precipitated proteins, organic solvent removal is performed on clear supernatants by three washings with large volumes of high-performance liquid chromatography (HPLC)-grade chloroform. The remaining aqueous phase, free of solvent and any lipid-soluble substances that may interfere with the further metabolite analysis, is used for the simultaneous ion-pairing HPLC determination of 39 compounds by means of a Kromasil C-18, 250 x 4.6-mm, 5-microm-particle-size column with tetrabutylammonium hydroxide as the pairing reagent. Results obtained by using the present method to prepare different rat tissue extracts demonstrate that it is possible to perform a single tissue preparation only for monitoring, in the same sample, compounds representative of the redox state (through the direct determination of GSH, GSSG, NAD+, NADH, NADP+, NADPH, CoASH, and oxidized CoASH) and of the cell energy state (by the analysis of oxypurines, nucleosides, and energy metabolites). Applicability of this sample processing procedure to quantify variations of the aforementioned compounds under pathological conditions was effected in rats subjected to moderate closed-head trauma.     

The three-dimensional structures of the Mycobacterium tuberculosis dihydrodipicolinate reductase-NADH-2,6-PDC and -NADPH-2,6-PDC complexes. Structural and mutagenic analysis of relaxed nucleotide specificity

Dihydrodipicolinate reductase (DHPR) catalyzes the reduced pyridine nucleotide-dependent reduction of the alpha,beta-unsaturated cyclic imine, dihydrodipicolinate, to generate tetrahydrodipicolinate. This enzyme catalyzes the second step in the bacterial biosynthetic pathway that generates meso-diaminopimelate, a component of bacterial cell walls, and the amino acid L-lysine. The Mycobacterium tuberculosis dapB-encoded DHPR has been cloned, expressed, purified, and crystallized in two ternary complexes with NADH or NADPH and the inhibitor 2,6-pyridinedicarboxylate (2,6-PDC). The structures have been solved using molecular replacement strategies, and the DHPR-NADH-2,6-PDC and DHPR-NADPH-2,6-PDC complexes have been refined against data to 2.3 and 2.5 A, respectively. The M. tuberculosis DHPR is a tetramer of identical subunits, with each subunit composed of two domains connected by two flexible hinge regions. The N-terminal domain binds pyridine nucleotide, while the C-terminal domain is involved in both tetramer formation and substrate/inhibitor binding. The M. tuberculosis DHPR uses NADH and NADPH with nearly equal efficiency based on V/K values. To probe the nature of this substrate specificity, we have generated two mutants, K9A and K11A, residues that are close to the 2'-phosphate of NADPH. These two mutants exhibit decreased specificity for NADPH by factors of 6- and 30-fold, respectively, but the K11A mutant exhibits 270% of WT activity using NADH. The highly conserved structure of the nucleotide fold may permit other enzyme's nucleotide specificity to be altered using similar mutagenic strategies.     

Effects of in vitro production on horse embryo morphology,cytoskeletal characteristics,and blastocyst capsule formation

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.     

A calcium-dependent bacterial surface protein is involved in the attachment of rhizobia to peanut roots

As part of a project to characterize molecules involved in the crack-entry infection process leading to nodule development, a microscopic assay was used to visualize the attachment of cells of Bradyrhizobium sp. strains SEMIA 6144 and TAL 1000 (labelled by introducing a plasmid expressing constitutively the green fluorescent protein GFP-S65T) to Arachis hypogaea L. (peanut). Qualitative and quantitative results revealed that attachment was strongly dependent on the growth phase of the bacteria. Optimal attachment occurred when bacteria were at the late log or early stationary phase. Cell surface proteins from the Bradyrhizobium sp. strains inhibited the attachment when supplied prior to the attachment assay. Root incubation with a 14-kDa protein (eluted from sodium dodecyl sulphate - gel electrophoresis of the cell surface fraction) prior to the attachment assay resulted in a strong decrease of attachment. The adhesin appeared to be a calcium-binding protein, since cells treated with EDTA were found to be able to bind to adhesin-treated peanut roots. Since this protein has properties identical to those reported for rhicadhesin, we propose that this adhesin is also involved in the attachment process of rhizobia to root legumes that are infected by the crack-entry process.     

Metabolic efficiency of liver mitochondria in rats with decreased thermogenesis

We have studied changes in hepatic mitochondrial efficiency induced by 24-h fasting or acclimation at 29 degrees C, two conditions of reduced thermogenesis. Basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, are not affected after 24-h fasting, when serum free triiodothyronine decreases significantly and serum free fatty acids increase significantly. In rats at 29 degrees C, in which serum free triiodothyronine and fatty acids decrease significantly, basal proton leak increases significantly, while no variation is found in palmitate-induced proton leak. The present results indicate that mitochondrial efficiency in the liver is not related to a physiological decrease in whole body thermogenesis.     

Methylation profile of P. lividus sea urchin genes during development

Methylation pattern has been studied in two genes of sea urchin Paracentrotus lividus using sodium bisulfite method to understand the possible role of DNA methylation during invertebrate development. Three regions of the gene for the hatching enzyme have been analyzed and all of them resulted unmethylated in embryos at different stages of development. Four CpG rich regions have been studied in the gene for DNA methyltransferase: upstream, upstream-exon1, intron 1 and exon 20. The upstream-exon 1 region is always unmethylated, while intron 1 and exon 20 are heavy methylated. Only the upstream fragment changed its pattern of methylation during development. For none of the studied regions the reported data show a general direct correlation between gene expression and methylation process during development.     

Inhibition of pig liver and Zea mays L. polyamine oxidase: a comparative study

Polyamine oxidase (PAO) is involved in polyamine metabolism and production of hydrogen peroxide in animal and plants, thus representing a key system in development and programmed cell death. In the present study, the inhibitory effect of amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenyl-indole (DAPI), gabexate mesylate, guazatine, and N,N'-bis(2,3-butadienyl)-1,4-butane-diamine (MDL72527) on the catalytic activity of pig liver and Zea mays L. PAO, Lens culinaris L. and Pisum sativum L. and swine kidney copper amine oxidase, bovine trypsin, as well as neuronal constitutive nitric oxide synthase (NOS-I) was investigated. Moreover, agmatine and N(3) -prenylagmatine (G3) were observed to inhibit pig liver and Zea mays L. PAO, bovine trypsin, and NOS-I action, but were substrates for Lens culinaris L., Pisum sativum L. and swine kidney copper amine oxidase. Guazatine and G3 inhibited selectively Zea mays L. PAO with K(i) values of 7.5 x 10(-9) M and 1.5 x 10(-8) M, respectively (at pH 6.5 and 25.0 degrees C). As a whole, the data reported here represent examples of enzyme cross-inhibition, and appear to be relevant in view of the use of cationic L-arginine-and imidazole-based compounds as drugs.