Use of behavioral and physiological indicators to evaluate Scaphirhynchus sturgeon spawning success

Thirty gravid, female shovelnose sturgeon (Scaphirhynchus platorynchus) were captured in the Lower Missouri River in March 2004 to evaluate the effectiveness of physiology, telemetry and remote sensor technology coupled with change point analysis in identifying when and where Scaphirhynchus sturgeon spawn. Captured sturgeons were instrumented with ultrasonic transmitters and with archival data storage tags (DST) that recorded temperature and pressure. Female sturgeon were tracked through the suspected spawning period. Thereafter, attempts were made to recapture fish to evaluate spawning success. At the time of transmitter implantation, blood and an ovarian biopsy were taken. Reproductive hormones and cortisol were measured in blood. Polarization indices and germinal vesicle breakdown were assessed on the biopsied oocytes to determine readiness to spawn. Behavioral data collected using telemetry and DST sensors were used to determine the direction and magnitude of possible spawning‐related movements and to identify the timing of potential spawning events. Upon recapture observations of the ovaries and blood chemistry provided measures of spawning success and comparative indicators to explain differences in observed behavior. Behavioral and physiological indicators of spawning interpreted along with environmental measures may assist in the determination of variables that may cue sturgeon reproduction and the conditions under which sturgeon successfully spawn.     

Use of charcoal to minimize end product inhibition in enzymatic hydrolysis of cellulose

Summary Tests made to improve saccharification of cellulose byTrichoderma cellulases showed that charcoal used as an adsorbent minimized the end product inhibition. Charcoal adsorbed both cellobiose and glucose and did not affect the enzymatic hydrolysis of cellulose. Results showed that charcoal is as effective as -glucosidase in improving the enzymatic saccharification of cellulose.     

Use of water-organic systems in the enzymatic transformation of steroids

This review surveys existing data on the use of water-organic systems, one of the promising methods of enzymic transformation of steroids. The procedures for reducing the inhibitory effect of solvents on enzymes and microbial cells are discussed. It is shown that hydrophobic capacity of gels and substrates, polarity of solvents, and coefficients of the distribution of reaction components between phases are to be taken into consideration when performing steroid transformation in the presence of organic solvents in order to increase its efficiency.     

Use of liposomes as membrane models to evaluate the contribution of drug-membrane interactions to antioxidant properties of etodolac

This work stresses the need to combine antioxidant assays and drug-membrane interaction studies to describe more accurately the antioxidant profile of non-steroidal anti-inflammatory drugs (NSAIDs). Different experiments performed in liposomes and aqueous solution were compared and used to evaluate the protective effect of etodolac in lipid peroxidation. Lipid peroxidation was induced by the peroxyl radical (ROO*) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydroxyl radical (HO*) generated by the Fenton reaction and was assessed by the fluorescence intensity decay of three fluorescence probes with distinct lipophilic properties--fluorescein; hexadecanoyl aminofluorescein (HDAF) and diphenylhexatriene propionic acid (DPHPA). Membrane fluidity changes due to lipid peroxidation were also evaluated by steady-state anisotropy measurements. Interactions of etodolac with lipid bilayers were evaluated by membrane zeta-potential measurements. Results indicate a drug location near the membrane surface and show that etodolac can scavenge the radicals studied but to a variable extent, depending on the assayed media and reactive species. The use of different probes and liposomes as membrane mimetic systems allowed us to conclude that membrane lipoperoxidation is not only related to the scavenging characteristics of the antioxidants, but also to their ability to interact with lipid bilayers.     

Comparison of enzymatic antioxidant defence systems in different metabolic types of yeasts

The enzymatic defence system in the 2 yeasts Kluyveromyces marxianus and Rhodotorula glutinis, differing in their mode of oxygen uptake and energy generation, was characterized and compared with the well-studied facultatively fermentative Crabtree-positive Saccharomyces cerevisiae strain. Twofold higher superoxide dismutase (SOD) and catalase activities were detected in K. marxianus and R. glutinis when cells were cultured on glucose. Further increases of 10%-15% in SOD activity and 30%-50% in catalase were measured in all studied yeasts strains after transfer to media containing ethanol. An evaluation of the ratio of Cu/Zn SOD / Mn SOD was performed as a measure of the oxidative metabolism. A 20% decrease was observed when the respiratory source of energy was ethanol, with the lowest ratio being observed for the oxidative type of K. marxianus yeasts. Electrophoretic analysis revealed that all tested strains possess active Cu/Zn and Mn SODs. A reverse electrophoretic mobility pattern of K. marxianus and R. glutinis SOD enzymes was observed in comparison with the same couple in S. cerevisiae. The investigation of electrophoretic profile of catalase enzymes showed that alongside their different taxonomic status and fermentative capacity, all tested strains possess 2 separate catalases. The role of antioxidant enzymes in preventing oxidant-induced cytotoxicity (treatment with hydrogen peroxide, paraquat, and menadione) was shown.     

Amplification of antioxidant activity and xanthine oxidase inhibition of catechin by enzymatic polymerization

To amplify the antioxidant activity, we synthesized poly(catechin) by the enzyme-catalyzed oxidative coupling using horseradish peroxidase as a catalyst. The poly(catechin) showed great improvement in antioxidant activity such as radical scavenging activity against the superoxide anion and inhibition effects against free radical induced-oxidation of low-density lipoprotein, compared with a catechin monomer. In addition, poly(catechin) showed very high inhibition effects on xanthine oxidase activity, whereas the catechin monomer showed very less inhibition effects. The amplified activities might offer a high potential as a therapeutic agent for prevention of various free radicals and/or enzyme-related diseases.     

Use of morphological and physiological characters, and molecular markers to evaluate the genetic diversity of three clementine cultivars

Originating from a natural crossing between mandarin and sweet orange at the end of the 19(th) century, clementine diversified through the selection of spontaneous mutations. Today, it seems almost impossible to distinguish one variety from another. The development of molecular tools for variety identification is thus necessary. Three clementine cultivars, representing distinct groups of fruit maturity, were evaluated. Identification criteria were searched at the phenotypical level (organoleptic characteristics, leaves morphology) as well as the DNA level (isozymes, RAPD, and ISSR). The phenotypical diversity observed is relatively high and contrasted with the low molecular polymorphism. In fact, only the cultivar 'Guerdane' presents profiles of genetic fingerprints different from those of the two other cultivars. The frequency of the genetic modifications would thus be variable from a cultivar to another. Moreover, the specific molecular markers of the cultivar 'Guerdane', added to the phenotypic markers, extend the possibilities of identification to the young nursery plants.     

Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.     

A comparison of methods employed to evaluate antioxidant capabilities

Three different methodologies frequently employed to evaluate the indexes that report the antioxidant capabilities of pure compounds and/or complex mixtures of antioxidants are applied to a series of mono- and polyphenols, as well as to two wine (red and white) samples. These methodologies are based on the bleaching of a stable radical, the effect of the additive upon luminol chemiluminescence induced by peroxyl radicals, and the effect of the additive upon the bleaching of the fluorescence from a dye molecule. Widely different responses are obtained from the different methodologies. These differences are interpreted in terms of the different factors (stoichiometric factors and/or reactivities) that determines the indexes evaluated by these different methodologies.     

A rapid microassay to evaluate enzymatic hydrolysis of lignocellulosic substrates

Current attempts to produce ethanol from lignocellulosic biomass are focused on the optimization of pretreatment to reduce substrate recalcitrance and the improvement of enzymes for hydrolysis of the cellulose and hemicellulose components to produce fermentable sugars. Research aimed at optimizing both aspects of the bioconversion process involves assessment of the effects of multiple variables on enzyme efficiency, resulting in large factorial experiments with intensive assay requirements. A rapid assay for lignocellulose hydrolysis has been developed to address this need. Pretreated lignocellulose is formed into handsheets, which are then used to prepare small disks that are easily dispensed into microtiter plates. The hydrolysis of cellulose to glucose is estimated using an enzyme-coupled spectrophotometric assay. Using disks prepared from ethanol organosolv pretreated yellow poplar, it is shown that the assay generates data comparable with those produced by hydrolysis of pretreated yellow poplar pulp in Erlenmeyer flasks, followed by HPLC analysis of glucose. The assay shows considerable time and cost benefits over the standard assay protocol and is applicable to a broad range of lignocellulosic substrates.     

A method to evaluate the antioxidant system for radicals in erythrocyte membranes

The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When an oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in erythrocyte membranes, using fatty acid spin labels with nitroxide radicals on the hydrocarbon chains. About 50 microL of packed (about 5-6 x 10(8)), carbon monoxide (CO)-gassed red blood cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37 degrees C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3 +/- 1.8 x 10(-3)/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occurring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.     

The nature of oxidants and antioxidant systems in the inhibition of mutation and cancer

We briefly review current concepts with regard to the nature of oxygen-derived oxidants in biological systems. Of these substances, hydroxyl radicals derived from hydrogen peroxide seem most likely to be involved in the various stages of carcinogenesis. Hydrogen peroxide detoxification, primarily through glutathione activity, is essential in preventing hydroxyl-radical formation. Transition metals such as iron play a central role in this latter process. Alterations in cellular macromolecules are most likely to take place if hydroxyl-radical formation is directed toward specific intramolecular sites by appropriately sequestered metals. For this reason, repair and turnover events are apt to be more important protective devices than are the actions of molecules which scavenge hydroxyl radicals. Although many cellular constituents are potential targets in free-radical and oxidant attacks leading to carcinogenesis, nucleic acids have been most extensively studied in this connection. On the basis of these investigations, it is a facile conclusion that oxidants might be involved in the early events of carcinogenesis as well as in transformation or promotion. The literature on antioxidants in chemoprevention in animals is supportive of such a role. However, other biochemical effects of antioxidants should raise a note of caution in the interpretation of animal experiments.     

Lipid peroxidation and antioxidant enzymatic systems in rat retina as a function of age

In the present study, we have assayed the enzymatic activity of Cu,Zn–SOD, Mn–SOD, GSH–Px, GSH-Red, Cat, and G6PD in rat retina as a function of age. Conjugated diene levels and MDA formation were also determined. The conjugated diene levels in rat retina were found to increase significantly with age, accompanied by a marked decrease in GSH–Px and Cat activities. No agerelated change in MDA levels and in GSH-Red and G6PD activity was found, whereas a significant increase in SOD activity was observed between 1 and 4 months. Decreased GSH–Px and Cat activity is related to increased lipid peroxidation with age.     

Activity of enzymatic antioxidant defense systems in chilled and heat shocked cucumber seedling radicles

Chilling whole cucumber seedlings that had 10‐mm long radicles for 4 days at 2.5°C significantly inhibited subsequent radicle growth both by increasing the time it took the seedlings to recover from chilling and attain a linear rate of radicle growth, and by decreasing the subsequent rate of linear growth. Exposing cucumber seedlings to 45°C for up to 20 min had no effect on subsequent radicle growth, while longer exposures produced reductions in growth. A heat shock at 45°C for 10 min induced the optimal protection to 4 days of chilling at 2.5°C by reducing chilling inhibition from 60 to 42%. Two hours after being chilled, heat shocked or heat shocked and then chilled, there was no difference in protein content of the apical 1 cm of the seedling radicle among these treatments and the non‐heat shocked, non‐chilled control. Two days after treatment, the protein content was still similar in tissue that had been heat shocked or heat shocked and chilled, while it was significantly reduced in tissue that had been chilled. In general, 2 h after treatment, the activity of the 5 antioxidant enzymes examined in this study [superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), guaiacol peroxidase (GPX; EC 1.11.1.7) and glutathione reductase (GR; EC 1.6.4.2)] were reduced by chilling and unaffected or increased by heat shock. When heat shock was followed by chilling, there was a consistent effect of the heat shock treatment on preventing the loss of enzyme activity following chilling. This protective effect of the heat shock treatment was even more pronounced after 2 days of recovery at 25°C for SOD, CAT and APX. In contrast, the activity of GR and GPX was substantially higher in chilled tissue than in tissue that had been heat shocked before being chilled. Elevated levels of GR and GPX therefore appear to be correlated with the development of chilling injury, while elevated levels of SOD, CAT and APX appear to be correlated with the development of heat shock‐induced chilling tolerance.     

Chemotaxis: the proper physiological response to evaluate phylogeny of signal molecules

In this review we summarize our results gained on the investigations focused to characterize ligand and signaling mechanisms required for the chemotaxis in the unicellular model Tetrahymena. Our data show that short chain signal molecules (amino acids, oligopeptides) are distinguished upon their physicochemical characteristics - lipophilicity, residual volumes and statistical distribution of side-chain distances (e.g. in proline containing dipeptides), while the vertebrate hormones have also specific attractant or repellent effects in the model (FSH vs. TSH). Hormonal imprinting developed by pretreatments has also special, signal molecule dependent effect (histamine vs. serotonin). It is shown that "chemotactic selection" of cells, by the new probe developed by us is a suitable tool to provide subpopulations possessing enhanced chemotactic receptor-effector mechanisms with respect to the selector signal molecules (IL-8, TNF-alpha).     

Role of the olfactory systems and importance of learning in the ewes' response to rams or their odors

In sheep, exposure of seasonally anestrous females to the male or its fleece results in activation of luteinizing hormone (LH) secretion and synchronized ovulation. The study of the neural pathways involved in this phenomenon, commonly named "male effect", show that the main olfactory system plays a critical role in the detection and the integration of the male odor. The accessory olfactory system participates in the perception of the ram odor but does not seem necessary for the endocrine response. According to the hypothesis that the neuroanatomical differences between the two olfactory systems could be associated with different functional roles, we investigated the importance of sexual experience and learning processes in the male effect. Our results showed that female responses depend on previous sexual experience. We also demonstrated that the LH response to male odor could result from an associative learning process. The aim of the present report was to summarize our current knowledge concerning the "male effect" and in particular to clarify the role of sexual experience and learning in the processes involved in this effect.