Effect of a ubiquinone-like molecule on oxidative energy metabolism in rat cortical synaptosomes at different ages

Persistent stimulation of energy consumption, induced by depolarization with veratridine, mimics a condition of abnormally enhanced energy demand and causes an increase in the oxygen consumption rate (QO₂) and in the interconversion of pyruvate dehydrogenase complex (PDHc) into its active form. Wistar rats at the age of 26 months do not show alterations of QO₂ and of the ability of veratridine to increase QO₂ in comparison with 6 month-old animals whereas the active form of PDHc is slightly but significantly reduced. Idebenone, a ubiquinone-like molecule (1 M), does not affect the QO₂ or PDHc activation state in resting conditions but attenuates the veratridine-challenged increase in QO₂ at all the ages tested and attenuates the increase in the percentage of PDHa reaching statistical significance in 26-month-old rats. At higher concentration (10 M) idebenone totally abolishes the veratridine-induced increase in PDHa also in the 6 month-old rats. At the lower concentration, the drug does not affect the increase in QO₂ induced by an uncoupler of oxidative phosphorylation. The results obtained suggest a protective effect of idebenone on the cerebral tissue against stressful conditions; this action may be exerted at the level of some mitochondrial component and/or on the Na⁺ homeostasis.     

Plasma lipoproteins, tissue cholesterol overload, and skeletal muscle apolipoprotein A-I synthesis in the developing chick

In the present study we investigated the changes of plasma lipids, lipoproteins, and tissue lipids that occur during the late embryonic life (5 days before hatching) and the postnatal period (0, 2, 7, 14, and 30 days after hatching) of the chick. The chick emerges from the egg with extreme hypercholesterolemia associated with a high level of cholesterol-rich VLDL + IDL. The density gradient profile of plasma lipoproteins showed that the concentrations of VLDL + IDL and LDL decreased during the first week of postnatal life, whereas HDL concentration increased sharply around hatching and remained stable afterwards. All plasma lipoprotein classes of the newborn chick (2 days from hatching) were enriched in cholesterol and cholesteryl esters; 2 weeks after hatching, the relative amount of cholesterol and cholesteryl esters decreased. In the newborn chick, plasma VLDL + IDL consisted of two populations of cholesteryl ester-rich lipoproteins: the main one (designated apoB-VLDL) contained apoB and no apoA-I; the other (designated apoA-I-VLDL) contained predominantly apoA-I. In the newborn chick there was an accumulation of free and esterified cholesterol in the liver and, to a lesser extent, in the skeletal muscle. These cholesterol deposits were depleted 2 to 7 days after hatching. The depletion in skeletal muscle was preceded by and associated with a striking increase in the synthesis of apoA-I in this tissue, as demonstrated by immunological methods and apoA-I mRNA measurements. In addition, apoA-I-containing HDL were secreted in vitro by explants of skeletal muscle of the newborn chick. The synthesis of apoA-I in the skeletal muscle decreased to the level found in the adult animal 1 week after hatching. It is likely that the rise of HDL and apoA-I in plasma observed 1-2 days after hatching reflects the production of apoA-I-containing HDL by skeletal muscle. We suggest that the cholesterol overload in skeletal muscle might stimulate the production of apoA-I which, in turn, would promote the removal of cholesterol from this tissue. The hypothesis that metabolic stimuli play a role in inducing apoA-I synthesis in skeletal muscle is supported by the observation that feeding the newborn chick a diet rich in proteins and lipids and free of carbohydrates delays the fall of apoA-I mRNA which normally occurs 1 week after hatching.     

Immunocytochemical evidence of vertebrate bioactive peptide-like molecules in the immuno cell types of the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata).

An immunocytochemical investigation was carried out on round and spreading hemocytes of Planorbarius corneus by using 20 antisera to vertebrate bioactive peptides. The immunotests showed the presence of alpha 1-antichymotrypsin-bombesin-, calcitonin-, CCK-8 (INC)-, CCK-39-, gastrin-, glucagon-, Met-enkephalin-, neurotensin-, oxytocin-, somatostatin-, substance P-, VIP-, and vasopressin-immunoreactive molecules in the spreading hemocytes. The round hemocytes were only positive to anti-bombesin, anticalcitonin, anti-CCK-8 (INC), anti-CCK-39, anti-neurotensin, anti-oxytocin, anti-substance P and anti-vasopressin antibodies. No immunostaining was observed with anti-CCK-8 (Peninsula), anti-insulin, anti-prolactin, anti-thyroglobulin and anti-thyroxin (T4) antibodies. As probably in vertebrates, these bioactive peptides may modulate immuno cell function.     

Three years of experience in biomanipulating a small eutrophic lake: Lago di Candia (Northern Italy)

Results obtained from a step by step approach to the biomanipulation of a natural lacustrine environment (Lago di Candia, Northern Italy) are presented. Since the diversion of the municipal sewage of the small town of Candia, runoff and precipitation have been the sole contributors of nutrient to the lake. Fish population is mainly characterized by rudd (Scardinius erythrophthalmus) overstocking and by a low density of large-mouth-bass (Micropterus salmoides) and pike (Esox lucius). During 1986 about 12t of rudd (1–2 year old) were removed from the lake. Considering 1986 as ‘control year’, average Secchi disc transparency improved from 2.3 m in 1986 to 3.3 m in 1988; phytoplankton biovolume decreased from 114 to 58 mm³ l⁻¹ but zooplankton biovolume increased from 8 to 11.5 mm³ l⁻¹. The results achieved show that a grodual biomanipulation treatment can have a satisfactory outcome, and has the advantage of not producing catastrophic situations either in the biotic or in the abiotic compartments of the lake.     

Identification of muramyl derivatives in mollusca gastropoda tissue

Summary Previous findings have demonstrated the presence of muramic acid and the lack of sialic acid in gastropod glycoconjugates from different tissues. The present study investigated the composition of muramyl derivatives in Mollusca Gastropoda tissue from the foot, mantle and periesophageal ganglia, using HRP-labeled lectins (LTA, UEA I, GSA IB₄, GSA II, DBA, SBA, RCA II, WGA, PNA, ConA) and glycosidase digestion (neuraminidase, lysozyme, -l-fucosidase, -N-acetylglucosaminidase, -N-acetylgalactosaminidase). Muramyl derivatives from the tissue examined showed some differences related to the composition of the terminal disaccharides. Indeed, foot and mantle mucocytes exhibited muramic acid in a terminal position, linked to (subterminal) N-acetylgalactosamine, whereas in neuron cells muramic acid was present in an internal position and linked to N-acetylglucosamine. Diversities also occurred between foot and mantle mucocytes with respect to the receptor sugar for penultimate N-acetylgalactosamine.     

The ability of the mitochondrial Ca2+-binding glycoprotein to restore Ca2+ transport in glycoprotein-depleted rat liver mitochondria

Rat liver mitochondria may be subfractionated in sediment and supernatant fractions by swelling in the presence of EDTA and oxaloacetate. The sediment is largely depleted of the Ca²⁺-binding glycoprotein and its Ca²⁺-transporting activity may be as low as 10–20% of the starting value. Both the rate of Ca²⁺ uptake and the capacity to maintain a high Ca²⁺ concentration gradient across the membrane are depressed. Addition of an osmotic supernatant to the assay mixture may partially restore the original Ca²⁺-transporting ability. The active component in the supernatant is the Ca²⁺-binding glycoprotein. This is shown by the following facts: (a) the effect is enhanced by the addition of the purified glycoprotein to the supernatant; (b) precipitation of the glycoprotein from the supernatant by affinity chromatography-purified antibodies abolishes the stimulatory effect, and (c) in the presence of 130 μM Mg²⁺, the glycoprotein alone may restore fully the Ca²⁺-transporting ability of the particles. The maximal velocity is already reached at 0.1 μg glycoprotein/mg mitochondrial protein.     

Predictive value of early virological response to treatment with different interferon-based regimens plus ribavirin in patients with chronic hepatitis C

Early virological response (EVR) to different interferon-based regimens plus ribavirin and its ability to predict the outcome of therapy in patients with chronic hepatitis C were investigated. The study design was as follows: 64 naive patients were considered, 32/64 received pegylated interferon alpha-2b (Peg-IFN-alpha2b) plus ribavirin and the remaining 32 received leucocyte interferon alpha (IFN-alpha) plus ribavirin. At week 4 of treatment, EVR was present in 68.7% and 37.5% of patients treated with Peg-IFN-alpha2b plus ribavirin, and with leucocyte interferon alpha (IFN-alpha) plus ribavirin, respectively (p = 0.024). At week 12, the cumulative EVR rates did not differ between the two groups (71.9% vs 56.2%, p >0.05) because a higher proportion of patients achieved EVR for the first time after more than 4 weeks of therapy in the standard IFN-alpha group. Sustained virological response (SVR) rates, however, resulted significantly higher in the Peg-IFN-alpha2b group (65.6% vs 37.5%; p = 0.045) since a higher proportion of patients who received standard IFN-alpha relapsed during the follow-up. In the standard IFN-alpha group, HCV genotype 1 (p = 0.035), high baseline viral load (p = 0.035) and the presence of bridging fibrosis/cirrhosis (p = 0.011) were closely associated with significantly lower SVR rates. In the Peg-IFN-alpha2b group, only bridging fibrosis/cirrhosis (p = 0.02) negatively influenced the outcome of treatment. Overall, 33/41 (80.5%) patients with EVR at week 12 were sustained responders, yielding a positive predictive value (PPV) of 0.80. However, when SVR was related to the time taken to reach EVR, 32/34 (94.1%) patients with EVR at week 4 of therapy (PPV = 0.94) versus 1/7 (14.3%) patients who had EVR after more than 4 weeks of therapy (PPV = 0.14) resulted sustained responders (p = 0.000057). In conclusion, EVR at week 4 of treatment is strongly associated with the likelihood of achieving SVR, regardless of the therapeutic regimen. However, when compared with standard IFN-alpha plus ribavirin, treatment with Peg-IFN-alpha2b plus ribavirin significantly increases the probability of viral clearance within the first 4 weeks of treatment. Finally, patients who do not clear the virus within the first 12 weeks of treatment have no chance of achieving SVR, justifying discontinuation of therapy in these patients.     

(1)H/(15)N heteronuclear NMR spectroscopy shows four dynamic domains for phospholamban reconstituted in dodecylphosphocholine micelles

We report the backbone dynamics of monomeric phospholamban in dodecylphosphocholine micelles using (1)H/(15)N heteronuclear NMR spectroscopy. Phospholamban is a 52-amino acid membrane protein that regulates Ca-ATPase in cardiac muscle. Phospholamban comprises three structural domains: a transmembrane domain from residues 22 to 52, a connecting loop from 17 to 21, and a cytoplasmic domain from 1 to 16 that is organized in an "L"-shaped structure where the transmembrane and the cytoplasmic domain form an angle of approximately 80 degrees (Zamoon et al., 2003; Mascioni et al., 2002). T(1), T(2), and (1)H/(15)N nuclear Overhauser effect values measured for the amide backbone resonances were interpreted using the model-free approach of Lipari and Szabo. The results point to the existence of four dynamic domains, revealing the overall plasticity of the cytoplasmic helix, the flexible loop, and part of the transmembrane domain (residues 22-30). In addition, using Carr-Purcell-Meiboom-Gill-based experiments, we have characterized phospholamban dynamics in the micros-ms timescale. We found that the majority of the residues in the cytoplasmic domain, the flexible loop, and the first ten residues of the transmembrane domain undergo dynamics in the micros-ms range, whereas minimal dynamics were detected for the transmembrane domain. Hydrogen/deuterium exchange factors measured at different temperatures support the existence of slow motion in both the loop and the cytoplasmic helix. We propose that these dynamic properties are critical factors in the biomolecular recognition of phospholamban by Ca-ATPase and other interacting proteins such as protein kinase A and protein phosphatase 1.     

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Cancer-germline genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.