José A. Lupiáñez

Localization of Nitric-oxide Synthase in Plant Peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca2+-dependent NOS activity of 5.61 nmol ofl-[3H]citrulline mg−1 protein min−1 was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60–90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.

Maslinic acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29 human colon-cancer cells via the mitochondrial apoptotic pathway

We have investigated the mechanisms of maslinic acid with regard to its inhibitory effects on the growth of HT29 colon-cancer cells. High concentrations of maslinic acid are present in the protective wax-like coating of olives. Our results show that treatment with maslinic acid results in a significant inhibition of cell proliferation in a dose-dependent manner and causes apoptotic death in colon-cancer cells. We found that it inhibits considerably the expression of Bcl-2 whilst increasing that of Bax; it also stimulates the release of mitochondrial cytochrome-c and activates caspase-9 and caspase-3. All these results point clearly to the activation of the mitochondrial apoptotic pathway in response to the treatment of HT29 colon-cancer cells with maslinic acid. Our results suggest that maslinic acid has the potential to provide significant natural defence against colon-cancer.

(2α,3β)-2,3-Dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells

Triterpenoids are known to induce apoptosis and to be anti‐tumoural. Maslinic acid, a pentacyclic triterpene, is present in high concentrations in olive pomace. This study examines the response of HT29 and Caco‐2 colon‐cancer cell lines to maslinic‐acid treatment. At concentrations inhibiting cell growth by 50–80% (IC50HT29 = 61 ± 1 μM, IC80HT29 = 76 ± 1 μM and IC50Caco‐2 = 85 ± 5 μM, IC80Caco‐2 = 116 ± 5 μM), maslinic acid induced strong G0/G1 cell‐cycle arrest and DNA fragmentation, and increased caspase‐3 activity. However, maslinic acid did not alter the cell cycle or induce apoptosis in the non‐tumoural intestine cell lines IEC‐6 and IEC‐18. Moreover, maslinic acid induced cell differentiation in colon adenocarcinoma cells. These findings support a role for maslinic acid as a tumour suppressant and as a possible new therapeutic tool for aberrant cell proliferation in the colon. In this report, we demonstrate for the first time that, in tumoural cancer cells, maslinic acid exerts a significant anti‐proliferation effect by inducing an apoptotic process characterized by caspase‐3 activation by a p53‐independent mechanism, which occurs via mitochondrial disturbances and cytochrome c release.

Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout. Adaptive response to starvation and a high-protein, carbohydrate-free diet on glutamate dehydrogenase and alanine aminotransferase kinetics

In fish, metabolic changes and qualitative responses during different nutritional situations are highly controversial in the scientific literature, and for this reason the objective of this work has been to probe deeper into the adaptive behaviour of two important amino acid-metabolising enzymes, glutamate dehydrogenase (GDH) and alanine aminotransferase (AAT) of liver and kidney in trout. In the present study, we examined the long-term effects of endogenous or exogenous proteins--generated, respectively, by a prolonged starvation or by feeding a high-protein diet--on the kinetics of liver and kidney GDH and AAT. Feeding on a high-protein diet significantly increased the liver (100%) and kidney (49%) GDH Vmax and catalytic efficiency; the same kinetic parameters of AAT increased by 65% only in the liver enzyme, without changing the Km and activity ratio values. Starvation registered a significant increase of both enzymes, Vmax and catalytic efficiency in the liver, but activity was unaltered in the kidney. In addition, no significant changes were found in the Km or activity ratio. All enzyme kinetics showed a Michaelian behaviour without any evidence of sigmoidicity. The experimental results show strong adaptive responses in the kinetic behaviour of the enzymes of both tissues. With the exception of renal AAT, the remainder of the enzymes presented a marked influence in their kinetic parameters by an excess of protein. The results are discussed in terms of the possible adaptive role of enzyme kinetics to amino acid availability.

Long-term effect of a high-protein/non-carbohydrate diet on the primary liver and kidney metabolism in rainbow trout (Salmo gairdneri)

Abstract Several aspects of the metabolic adaptation of trout liver and kidney to a high-protein diet were studied. Under this nutritional situation there were significant increases in the activities of the glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK) from both tissues but with a different molecular basis. PFK activity was increased at subsaturating substrate concentrations, with a decrease in Michaelis constant (Km) values of about 70% in both tissues and without changes in the maximum velocity (Vmax). The activity of PK was increased all along the saturation curve, approximately 70% and 20% in liver and kidney respectively. However, no changes in the activities of glucose 6-phospate dehydrogenase (G6PDH) were detected. On the other hand, the activity of the gluconeogenic enzyme fructose 1,6-bisphosphatase (FBPase) from both tissues was increased only at cellular substrate concentrations. With respect to amino acid metabolism, liver alanine aminotransferase (AAT) was enhanced at all substrate concentrations, with a significant increase of almost 50% in the Vmax, without changes in the renal activity. Glutamate dehydrogenase (GDH) activity was almost 300% and 100% of control values, in liver and kidney respectively, observations which are in good agreement with the role of this enzyme in the two tissues.

Carbohydrates affect protein-turnover rates, growth, and nucleic acid content in the white muscle of rainbow trout (Oncorhynchus mykiss)

Abstract We have investigated the effect of dietary carbohydrate on different parameters of protein-turnover rate, nature of growth, and nucleic acid content in the muscle of rainbow trout in order to better understand the molecular nature of these growth parameters in the absence of this dietary component. For this, we used a methodology based on the incorporation rate of tritium labelled phenylalanine in muscle protein. Juvenile rainbow trout of an initial body weight of 110 g were fed near to satiety with a control or a non-carbohydrate diet during 7 weeks. The absence of dietary carbohydrate significantly depressed fish growth, as well as daily body weight gain, as a consequence of muscular hypotrophy (the cell size diminished by almost 50%) and not by a reduction of number of cells (hypoplasia). This nutritional situation also significantly slowed (by almost 11%) muscle-protein accumulation rate ( K G ) as a result of a significant increase (eight-fold) in muscle-protein degradation rate ( K D ), without changing the other protein-turnover rates, protein synthesis rate ( K S ), protein synthesis capacity ( C S ), protein synthesis efficiency ( K RNA ), protein synthesis rate per cell unit ( K DNA ), or protein retention efficiency (PRE). These results, together with the nucleic acid content, clearly indicate that the absence of carbohydrate significantly exacerbates the muscular-protein degradation without affecting protein synthesis. In conclusion, carbohydrates are needed to prevent amino acids released during protein degradation from being used to synthesize carbohydrates and/or to be used for energy and not for growth.

Impact of starvation-refeeding on kinetics and protein expression of trout liver NADPH-production systems

Herein we report on the kinetic and protein expression of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase, and malic enzyme (ME) in the liver of the trout (Oncorhynchus mykiss) during a long-term starvation-refeeding cycle. Starvation significantly depressed the activity of these enzymes by almost 60%, without changing the Michaelis constant. The time response to this nutritional stimulus increased with fish weight. The sharp decline in G6PDH and ME activities was due to a specific protein-repression phenomenon, as demonstrated by molecular and immunohistochemical analyses. Also, the dimeric banding pattern of liver G6PDH shifted from the fully reduced and partially oxidized forms, predominant in control, to a fully oxidized form, more sensitive to proteolytic inactivation. Refeeding caused opposite effects in both protein concentration and enzyme activities of about twice the control values in the first stages, later reaching the normal enzyme activity levels. Additionally, the partially oxidized form of G6PDH increased. The kinetics of these enzymes were examined in relation to the various metabolic roles of NADPH. These results clearly indicate that trout liver undergoes protein repression-induction processes under these two contrasting nutritional conditions.

Liver and white muscle protein turnover rates in the / European eel Anguilla anguilla: effects of dietary protein quality

Abstract The influence of the quality of dietary protein source on growth and protein synthesis and degradation rates was studied in the liver and white muscle of the European eel. Fish were fed isonitrogenous diets differing in protein source: one (control) contained fish meal, three others incorporating meat meal (MM) or sunflower meal (SFM) as the only protein source, and SFM supplemented with some essential amino acids (EAAs) were also tested. Fish fed diets containing unsupplemented MM or SFM exhibited dietary utilization and growth indices poorer than those fed the control, while EAA supplementation greatly improved the performance of the SFM-diet. Liver showed higher rates of protein synthesis ( k s ) and degradation ( k d ) associated with a higher capacity for protein synthesis per unit of DNA but a lower protein deposition efficiency (PDE), compared to muscle. Low quality dietary protein increased the protein turnover rate, with a higher protein synthesis rate per unit of DNA and RNA but a decrease of PDE. In white muscle, MM and unsupplemented SFM diets decreased k s without changing k d . The MM diet reduced the efficiency of protein synthesis and deposition. EAA supplementation of the SFM diet raised the protein synthesis rate and capacity as well as protein deposition compared to control values.

Growth, protein-turnover rates and nucleic-acid concentrations in the white muscle of rainbow trout during development.

We have studied the growth rate, nucleic-acid concentration, protein-accumulation rate (K(G)), and several other parameters relating to protein turnover, such as the protein-synthesis (K(S)), and protein-degradation rates (K(D)), protein-synthesis capacity (C(S)), protein-synthesis efficiency (K(RNA)), protein-synthesis rate per DNA unit (K(DNA)) and protein-retention efficiency (PRE), in the white muscle of rainbow trout during development. Both growth rate and relative food intake decreased significantly with age and weight, as did the food-efficiency ratio (FER) and protein-efficiency ratio (PER). Although absolute RNA and DNA contents increased with age, their relative concentrations decreased. The RNA/DNA ratio increased sharply from 14 to 28 weeks but afterwards decreased towards initial values. Hypertrophy increased rapidly to the 28-week stage but henceforth increased much more slowly. Hyperplasia, on the other hand, continued to increase linearly, resulting in a significant four- to fivefold predominance in this type of growth at the end of the 96-week experimental period. K(G) decreased significantly with age, as did K(S), and C(S), whereas at the 14-week stage, K(D) was significantly lower than at other ages. K(RNA) increased until 28 weeks. K(DNA) increased significantly in juvenile fish compared to both fingerlings and adults, where it showed similar lower values. PRE remained high at all ages.

Kinetic properties of hexose-monophosphate dehydrogenases. I. Isolation and partial purification of glucose-6-phosphate dehydrogenase from rat liver and kidney cortex☆

Glucose-6-phosphate dehydrogenase (G6PDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely separated from 6-phosphogluconate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 40-55% ammonium sulphate fractionation, by which both enzyme activities were separated, and finally, the application of the protein fraction to a column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of isolated liver and renal G6PDH were examined. Both enzymes showed a typical Michaelis-Menten kinetic saturation curve with no evidence of co-operativity. The optimum pH of both liver and kidney cortex G6PDH was 9.4. The Km values for glucose-6-phosphate (G6P) and for NADP were 3.29 x 10(-4) M and 1.00 x 10(-4) M respectively. The specific activity measured at 37 degrees C and optimum pH was 327.1 mU/ mg of protein. NADPH caused a competitive inhibition with a Ki of 10 microM. The Km values for the G6P and NADP of kidney-cortex G6PDH were 2.06 x 10(-4) and 0.25 x 10(-4) M respectively. The specific activity at pH 9.4 and 37 degrees C was 76.55 mU/mg of protein. The Ki value for NADPH inhibition was 4 microM. This work describes an easy, rapid and reliable method for the separation of the two dehydrogenases involved in the hexose-monophosphate shunt in animal tissues.