Metabolic Profiling of Alternative NAD Biosynthetic Routes in Mouse Tissues

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the “amidated” and “deamidated” routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes''rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme''s substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.     

Endothelial cells as early sensors of pulmonary interstitial edema.

We studied responses of endothelial and epithelial cells in the thin portion of the air-blood barrier to a rise in interstitial pressure caused by an increase in extravascular water (interstitial edema) obtained in anesthetized rabbits receiving saline infusion (0.5 ml.kg(-1).min(-1) for 3 h). We obtained morphometric analyses of the cells and of their microenvironment (electron microscopy); furthermore, we also studied in lung tissue extracts the biochemical alterations of proteins responsible for signal transduction (PKC, caveolin-1) and cell-cell adhesion (CD31) and of proteins involved in membrane-to-cytoskeleton linkage (alpha-tubulin and beta-tubulin). In endothelial cells, we observed a folding of the plasma membrane with an increase in cell surface area, a doubling of plasmalemma vesicular density, and an increase in cell volume. Minor morphological changes were observed in epithelial cells. Edema did not affect the total plasmalemma amount of PKC, beta-tubulin, and caveolin-1, but alpha-tubulin and CD-31 increased. In edema, the distribution of these proteins changed between the detergent-resistant fraction of the plasma membrane (DRF, lipid microdomains) and the rest of the plasma membrane [high-density fractions (HDFs)]. PKC and tubulin isoforms shifted from the DRF to HDFs in edema, whereas caveolin-1 increased in DRF at the expense of a decrease in phosphorylated caveolin-1. The changes in cellular morphology and in plasma membrane composition suggest an early endothelial response to mechanical stimuli arising at the interstitial level subsequently to a modest (approximately 5%) increase in extravascular water.     

In vivo and in silico anti-inflammatory mechanism of action of the semisynthetic (−)-cubebin derivatives (−)-hinokinin and (−)-O-benzylcubebin

(?)-Cubebin (CUB), isolated from seeds of Piper cubeba, was used as starting material to obtain the derivatives (?)-hinokinin (HK) and (?)-O-benzyl cubebin (OBZ). Using paw edema as the experimental model and different chemical mediators (prostaglandin and dextran), it was observed that both derivatives were active in comparison with both negative (5% Tween® 80 in saline) and positive (indomethacin) controls. The highest reduction in the prostaglandin-induced edema was achieved by OBZ (66.0%), while HK caused a 59.2% reduction. Nonetheless, the dextran-induced paw edema was not significantly reduced by either of the derivatives (HK or OBZ), which inhibited edema formation by 18.3% and 3.5%, respectively, in contrast with the positive control, cyproheptadine, which reduced the edema by 56.0%. The docking analysis showed that OBZ presented the most stable ligand-receptor (COX-2 – cyclooxygenase-2) interaction in comparison with CUB and HK.     

A potential role of alkaloid extracts from Salsola species (Chenopodiaceae) in the treatment of Alzheimer's disease

From the aerial parts of Salsola oppositofolia, S. soda and S. tragus an alkaloid extract was obtained and tested to evaluate antioxidant and anti-cholinesterase activities. The in vitro study of the antioxidant activity by the DPPH method revealed a significant activity of Salsola alkaloid extracts with IC₅₀ values ranging from 16.30 μg/mL for S. oppositifolia to 26.17 μg/mL for S. tragus. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were evaluated. S. tragus alkaloid extract exerted the highest inhibitory activity against AChE (IC₅₀ of 30.2 μg/mL) and BChE (IC₅₀ of 26.5 μg/mL). Interestingly, S. soda and S. oppositifolia exhibited a selective inhibitory activity against BChE with IC₅₀ values of 34.3 μg/mL and 32.7 μg/mL, respectively. Tetrahydroisoquinoline alkaloids were identified and quantified by GC/MS analysis.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.