Physiology of lipase formation inPenicillium candidum

Penicillium candidum grew and produced lipase in a culture medium supplemented with 0.2% olive oil. Significant enzyme production required the presence of olive, oil and was prevented by cycloheximide. Polyacrylamide gel electrophoresis of filtrates from olive oil fermentations gave a single band of lipase activity (MW 80 KDa). Among the olive oil components only oleate allowed significant lipase production. Other carboxylic and saturated fatty acids containing similar or lower numbers of carbon atoms, did not cause derepression of lipase formation.     

Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae

Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO₄ ^3- concentration and the growth was completely arrested at a concentration of 20 mM of VO₄ ^3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A mobile and an immobile species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time _r indicated the relative motional freedom at the macromolecular site. A strongly immobilized vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO²⁺ ions with isolated cell walls.     

Single-cell investigation by laser scanning confocal microscopy of cytochemical alterations resulting from extracellular oxidant challenge

Confocal laser scanning fluorescence microscopy coupled to image analysis was employed in order to develop and evaluate procedures for the appraisal at the single-cell level of: (1) protein-bound 4-hydroxynonenal, the specific product of membrane peroxidation (by means of immunocytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2,4-dinitrophenyl-hydrazine followed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol-specific fluorescent reagents possessing different cell permeabilities). The procedures proved able to reveal the subcellular distribution of cytochemical parameters useful as indices of oxidative stress conditions, and may allow redox phenotyping of isolated cells, which would provide an efficient tool in selected experimental models.     

Selective colocalization of lipid peroxidation and protein thiol loss in chemically induced hepatic preneoplastic lesions: the role of γ-glutamyltranspeptidase activity

A number of studies indicate that cell proliferation can be modulated by changes in the redox balance of (soluble and protein) cellular thiols. Free radical processes, including lipid peroxidation (LPO), can affect such a balance, and a role for LPO in multistage carcinogenesis has been envisaged. The present study was aimed to assess the relationships between the protein thiol redox status and the LPO process in chemically induced preneoplastic tissue. The Solt-Farber's initiation-promotion model of chemical carcinogenesis in the rat liver was used. In fresh cryostat sections, preneoplastic lesions were identified by the reexpression of γ-glutamyltranspeptidase (GGT) activity. In serial sections, different classes of protein thiols were stained; in additional sections, LPO was elicited by various prooxidant mixtures and determined thereafter by the hydroxynaphthoic hydrazide-Fast Blue B procedure. The incubation of sections in the presence of chelated iron plus substrates for GGT activity leads to the development of LPO in selected section areas closely corresponding to GGT-positive lesions, indicating the ability of GGT activity to initiate LPO. Protein-reactive thiols, as well as total protein sulfur, were decreased by 20–25% in cells belonging to GGT-positive preneoplastic nodules, suggesting the occurrence of oxidative conditions in vivo. The incubation of additional adjacent sections with the prooxidant mixture H₂O₂ plus iron(II), in order to induce the complete oxidation of lipid present in the section, showed a decreased basal concentration of oxidizable lipid substrate in GGT-rich areas. The decreased levels of both protein thiols and lipid-oxidizable substrate in GGT-positive nodules suggest that the observed GGT-dependent path-way of LPO initiation can be chronically operative in vivo during early stages of chemical carcinogenesis, in cells expressing GGT as part of their transformed phenotype.     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

Circadian and seasonal variation of the body temperature of sheep in a tropical environment

Nychthemeral and annual rhythms of the rectal temperature were determined for Corriedale sheep in a tropical climate. The minimum rectal temperature averaged 39.55° C at 0500 hours in summer, and 38.87° C at 0600 hours in winter. The maximum was 40.03° C in summer (1700 hours) and 39.33° C in winter (1830 hours). Annual cycle of the rectal temperature showed a minimum in July and maximum in December.     

Fluctuating temperatures have different effects on embryonic sensitivity to ABA in Sorghum varieties with contrasting pre-harvest sprouting susceptibility

The effect of fluctuating temperatures on the germination ofimmature caryopses of two Sorghum varieties presenting contrastingsusceptibility to pre-harvest sprouting was investigated. Fluctuatingtemperatures were able to stimulate germination of immaturecaryopses of both varieties from early stages of development(i.e. 15 d after pollination). Isolated embryos from both varietiesgerminated well in water irrespective of the thermal regimeof incubation. However, the ability of ABA to block germinationin Redland B2 (sproutingsusceptible) isolated embryos was significantlyreduced when embryos were incubated under fluctuating temperaturesfrom 23 DAP onwards. No such effect was found in IS 9530 (sprouting-resistant)embryos. No differences in the pattern with which embryonicABA content decreased during whole grain incubation were foundin 25 and 35 DAP grains from both varieties incubated underconstant or fluctuating temperatures. Therefore, these resultsindicate that alternating temperatures can promote germinationthrough different mechanisms. One of them is the decrease inembryo sensitivity to ABA inhibition which appears to be actingin Redland B2 caryopses from 23 DAP onwards; the other one seemsto be independent of ABA level and sensitivity and is activeat very early stages of development in one variety (RedlandB2) and throughout seed development in the other (IS 9530). Key words: Germination, dormancy, fluctuating temperatures, abscisic acid, seed development, Sorghum bicolor     

Toxicology of vanadium compounds in diabetic rats: The action of chelating agents on vanadium accumulation

The possible use of vanadium compounds in the treatment of diabetic patients is now being evaluated. However, previously to establish the optimal maximum dose for diabetes therapy, it should be taken into account that vanadium is a highly toxic element to man and animals. The toxic effects of vanadium are here reviewed. The tissue vanadium accumulation, which would mean an additional risk of toxicity following prolonged vanadium administration is also discussed. Recently, it has been shown that coadministration of vanadate and TIRON, an effective chelator in the treatment of vanadium intoxication, reduced the tissue accumulation of this element, decreasing the possibility of toxic side effects derived from chronic vanadium administration without diminishing the hypoglycemic effect of vanadium. However, previously to assess the effectiveness of this treatment in diabetic patients, a critical reevaluation of the antidiabetic action of vanadium and its potential toxicity is clearly needed.     

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997