Magic angle spinning 13C-NMR spin-lattice relaxation study of the location and effects of α-tocopherol,ubiquinone-10 and ubiquinol-10 in unsonicated model membranes

-Tocopherol, ubiquinone-10 and ubiquinol-10 have been studied by high resolution magic angle samples spinning ¹³C-nuclear magnetic resonance in egg yolk phosphatidylcholine multilamellar vesicles model membranes in order to assess their location and the induced perturbations on this model system. -Tocopherol is placed in such a position that it is in close contact with the head group of the phospholipid and exposed to the solvent. In this position it significantly perturbs the phospholipid head group, the 5a-CH₃ and the 7a-CH₃ groups being the closest parts to the membrane surface. On the other hand, ubiquinol-10 perturbs the membrane surface more than ubiquinone-10, but neither compound significantly changed the phospholipid head group conformation.     

Induced spawning by LHRHa and pimozide in the Asian catfish Clarias macrocephalus (Gunther)

Experiments were conducted to determine the optimum dose of luteinizing hormone-releasing hormone analogue (LHRHa) and pimozide (PIM) injected simultaneously to yield a high ovulation rate and produce sufficient eggs in the Asian catfish Clarias macrocephalus . In June 1990, injection of 0.05 or 0.10 μg LHRHa/g body weight (BW) + 1 μg PIM/g resulted in 100% ovulation, while only 80% of gravid catfish injected 0.025 μg LHRHa + 1 μg PIM/g ovulated. Most of the eggs stripped from 6 out of 8 control fish were not mature. Fertilization and hatching rates of LHRHa + PIM-induced fish (75–90% and 39–51%, respectively) were higher than those of control fish (36–39% and 0–1% respectively). In August and September 1990, at gravid catfish ovulated after injection of 0.05–0.10 μg LHRHa + 1 μg PIM/g BW. However, only 20% of the fish given 0.025 μg LHRHa/g + 1 μg PIM/g BW in August ovulated. No eggs could be striped from any of the control fish in August and September 1990. Techniques developed in this study, showed a simple and effective way of spawning captive catfish, C. macrocephalus . A simultaneous intramuscular injection of 0.05 μg LHRHa + 1 μg PIM/g and stripping of eggs at 16–20 h post-injection have been tested to yield high ovulation, fertilization and hatching rates.     

Life history and reproductive potential of the agarophyte Gelidium robustum in California

The reproductive potential of the tetrasporangial phase of Gelidium robustum was studied for 16 months at two sites off Santa Barbara, California. In all samples tetrasporangial thalli were always more abundant than gametangial ones. Tetratrasporangial sori were present throughout the duration of the study but relative fecundity was highest [300–400 sori g^–1 (w. wt)] in spring/summer samples of consecutive years, as a result of increasing numbers both of tetrasporangial branchlets per plant and of sori per branchlet. On the other hand, laboratory experiments showed that tetraspore release per sorus was highest (150–250 spores sorus^–1 d^–1) in winter. Inferring from these field and laboratory data plants released up to ± 34 000 tetraspores g^–1 (w. wt) d^–1 in the spring/summer of the second study year. Tetraspore germination, under defined culture conditions, also showed a marked seasonality increasing sharply from less than 10% in winter up to almost 60% in spring/summer, thus coinciding with the period of maximal spore output per plant. These results suggest that although relatively high numbers of tetraspores may be released by G. robustum plants all year round these might not always have the potential to germinate and recruit.     

Revealing the polar lipidome,pigment profiles,and antioxidant activity of the giant unicellular green alga,Acetabularia acetabulum

Marine algae are one of the most important sources of high-value compounds such as polar lipids, omega-3 fatty acids, photosynthetic pigments, or secondary metabolites with interesting features for different niche markets. Acetabularia acetabulum is a macroscopic green single-celled alga, with a single nucleus hosted in the rhizoid. This alga is one of the most studied dasycladalean species and represents an important model system in cell biology studies. However, its lipidome and pigment profile have been overlooked. Total lipid extracts were analyzed using hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS), tandem mass spectrometry (MS/MS), and high-performance liquid chromatography (HPLC). The antioxidant capacity of lipid extracts was tested using DPPH and ABTS assays. Lipidomics identified 16 polar lipid classes, corresponding to glycolipids, betaine lipids, phospholipids, and sphingolipids, with a total of 191 lipid species, some of them recognized by their bioactivities. The most abundant polar lipids were glycolipids. Lipid classes less studied in algae were identified, such as diacylglyceryl-carboxyhydroxymethylcholine (DGCC) or hexosylceramide (HexCer). The pigment profile of A. acetabulum comprised carotenoids (17.19%), namely cis-neoxanthin, violaxanthin, lutein and β,β-carotene, and chlorophylls a and b (82.81%). A. acetabulum lipid extracts showed high antioxidant activity promoting a 50% inhibition (IC₅₀) with concentrations of 57.91 ± 1.20 μg · mL⁻¹ (438.18 ± 8.95 μmol Trolox · g⁻¹ lipid) in DPPH and 20.55 ± 0.60 μg · mL⁻¹ in ABTS assays (918.56 ± 27.55 μmol Trolox · g⁻¹ lipid). This study demonstrates the potential of A. acetabulum as a source of natural bioactive molecules and antioxidant compounds.     

Liquid-liquid extraction of a recombinant cutinase from fermentation media with AOT reversed micelles

This work reports the extraction and back-extraction of an intracellular recombinant cutinase from complex biological media using AOT reversed micelles in isooctane. Cutinase was recovered from different complex media namely, fermentation broths and supernatants after cell disruption by osmotic shock and sonication. The application of the AOT reversed micellar system to the extraction of cutinase allowed activity yields and purification factors ranging from about 5% to 50% and 1.2 to 10.2, respectively, depending on the biological medium.Maria das Graças Carneiro da Cunha, from ITEP-Instituto Tecnológico do Estado de Pernambuco, acknowledges a Ph.D fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Centro de Pesquisa Aggeu Magalhães, Recife — PE — Brasil. E. P. Melo thanks Junta Nacional de Investigação Científica, Lisboa, Portugal, for providing a Ph.D. fellowship. The scientific support given by Prof. Sílvia M. B. Costa for the spectroscopic data and further discussions are particularly acknowledged.This work was partly financed by the BRIDGE and BIOTECHNOLOGY Programmes (Contracts BIOT-CT91-0274(DTEE) and BIOT 2 CT-943016).     

Effects of anti-inflammatory drugs on fever and neutrophilia induced by Clostridium difficile toxin B

This study investigated the ability of Clostridium difficile toxin B, isolated from the VPI 10463 strain, to induce fever and neutrophilia in rats. Intravenous injection of toxin B (0.005-0.5 mug/kg) evoked a dose-dependent increase in body temperature. The febrile response to 0.5 mug/kg of the toxin started in 2.5 h, peaked at 5 h, and subsided fully within 24 h. Toxin B also induced a dosedependent neutrophilia. Pretreatment with indomethacin (2 mg/kg, i.p.) did not affect the neutrophilia induced by toxin B, but significantly reduced the febrile response measured 4 to 8 h after toxin B injection. Dexamethasone (0.5 mg/ kg) also markedly diminished the febrile response induced by toxin B. These results show that Clostridium difficile toxin B induced a febrile response susceptible to inhibition by dexamethasone and indomethacin. Furthermore, they suggest that prostaglandins are not involved in the neutrophilia caused by this toxin.     

Constitutive expression of small heat shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum

Using antibodies raised against two sunflower small heat shock proteins (sHSPs), we have detected immunologically related proteins in unstressed vegetative tissues from the resurrection plant Craterostigma plantagineum. In whole plants, further accumulation of these polypeptides was induced by heat-shock or water-stress. In desiccation-intolerant Craterostigma callus tissue, we failed to detect sHSP-related polypeptides, but their expression, and the concurrent acquisition of desiccation tolerance was induced by exogenous abscisic acid (ABA) treatment. In untressed plants, the cross-reacting polypeptides were abundant in the roots and lower part of the shoots, where they showed homogeneous tissue-distributions. This constitutive expression is novel for vegetative tissues of higher plants, and resembles the expression patterns of sHSPs in desiccation-tolerant zygotic embryos and germinating seeds.J.A. and C.A. contributed equally to this work and are both considered to be first author     

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions

In order to elucidate the relationship between hypertension and hypertrophy in the production of heat shock proteins, we studied the induction of the HSP72 synthesis by the heart and gracilis muscles of normo (WKY) and hypertensive (SHR) rats subjected to hyperthermia (42°C±0.5 for 15 min). Two age groups were investigated in each strain: young (2 months, with developing cardiac hypertrophy) and old (18 months, with fully developed chronic cardiac hypertrophy). The gracilis muscle never developed hypertrophy, independently of hypertension or aging. 72 kDa inducible protein was determined by Western blot analysis using a specific monoclonal antibody. We also used a commercial standard, loaded on each blot, to quantitate densitometrically the signal.The heart of young SHR responds to heat shock more than their normotensive age-matched control (298.8±24.7% vs 88.3 ±8.5%, p<0.001). This response is not maintained during aging as we did not find any significant difference between normo-and hypertensive old rats after exposure to hyperthermia (43.6±5.3% vs 65.3±10.4%).Unlike the heart, the gracilis muscle shows a basal spontaneous HSP72 synthesis in both the SHR (71.4±10.8%) and WKY (40.6±11.7%) animals. There was a significant increase in HSP72 synthesis in the gracilis muscle of young SHR with respect to their control (186.2±18.7% vs 115.8±9.9%, p<0.02) which was maintained also during aging (171.9±17.3% vs 95.2±10.5%, p<0.01).In conclusion, these data show that hypertension results in an increased synthesis of HSP72 both in cardiac and gracilis muscle in response to heat shock. This abnormal response is attenuated by aging in the heart but not in the gracilis muscle. Thus, the abnormality seems to be independent from hypertrophy and linked to genetic determination of the disease.     

Identification and Characterization of a New Serotonergic Recognition Site with High Affinity for 5-Carboxamidotryptamine in Mammalian Brain

Abstract: We analyzed the existence of an additional serotonin (5-HT) receptor subtype, sensitive to 5-carboxamidotryptamine, in the mammalian brain. Radioligand binding studies with [³H]5-HT were carried out in rat, guinea pig, and human brain membranes, in the presence of unlabeled drugs to mask the binding to all known 5-HT receptors, with the exception of 5-HT_1E sites. Under these conditions, unlabeled 5-carboxamidotryptamine still showed a biphasic competition curve with a nanomolar affinity component. Saturation studies with 5-[³H]carboxamidotryptamine were carried out in the presence of (±)-8-hydroxy-2-(di- n -propylamino)tetralin, mesulergine, and ergotamine, to mask the binding to all receptors known to be labeled by 5-carboxamidotryptamine. These studies showed the existence in cortex and hippocampus from guinea pig and human brain of a remaining binding site with high affinity ( pK _D = 7.8–8.1) and a unique pharmacological profile. 5-HT and 5-carboxamidotryptamine showed nanomolar affinity, whereas 5-methoxytryptamine recognized this binding site with intermediate affinity. Other drugs exhibited low or very low potency in inhibiting this binding. The addition of 5'-guanylylimidodiphosphate significantly reduced the number of binding sites labeled by 5-[³H]carboxamidotryptamine, in the presence of the masking drugs described above, indicating the interaction with a GTP-binding protein. Preliminary autoradiographic studies in human brain appear to indicate that this 5-HT binding site is present in areas such as the globus pallidus, neocortex, and hippocampus, among others.